Methods of treating cancer using lsd1 inhibitors in combination with immunotherapy

ABSTRACT

Provided herein are methods of treating cancer using LSD1 inhibitors in combination with immunotherapy.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.16/758,474, filed Apr. 23, 2020, which is a U.S. National PhaseApplication under 35 U.S.C. § 371 of International Application No.PCT/US2018/057058, filed on Oct. 23, 2018, which claims priority to U.S.Provisional Patent Application Ser. No. 62/576,001, filed Oct. 23, 2017,and U.S. Provisional Patent Application Ser. No. 62/688,002, filed Jun.21, 2018; the entire contents of each of which are herein incorporatedby reference.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

This invention was made with Government support under Grant Nos.CA118487 and CA210104 awarded by the National Institutes of Health. TheGovernment has certain rights in the invention.

SEQUENCE LISTING

This application contains a Sequence Listing that has been submittedelectronically as an XML file named 37314-0064002_SL_ST26.xml and ishereby incorporated by reference in its entirety. The XML file, createdon May 16, 2023, is 144,552 bytes in size.

TECHNICAL FIELD

The present invention relates to the treatment of cancer.

BACKGROUND

Chromatin modifications play a broad and general role in regulating geneexpression, and when they go awry, can lead to diseases. Consistent withthis notion, recent cancer genome sequencing efforts have identifiedmutations in chromatin regulators, and in the case of hematopoieticcancers, chromatin regulators are one of the most frequently mutatedgroups of genes.

SUMMARY

Without wishing to be bound by theory, the present results provideevidence that the histone H3K4 demethylase, lysine-specific demethylase1A (LSD1, also known as KDM1A) plays a critical role in suppressingendogenous double stranded RNA (dsRNA) levels and interferon responsesin tumor cells, by regulating transcription of endogenous retroviralelements (ERVs) and dsRNA turnover mediated by the RNA-inducingsilencing complex (RISC). dsRNA stress can lead to increased T cellinfiltration and an enhanced anti-tumor T cell immunity to transplantedtumors cells lacking LSD1, as these tumors showed significant growthdisadvantage only in the immunocompetent mice. Furthermore, depletion ofLSD1 rendered programmed cell death 1 (PD-1) blockade-refractory B16tumors significantly responsive to anti-PD-1 therapy. Collectively, LSD1was identified as a critical regulator of anti-tumor immunity, therebysuggesting that manipulating LSD1 can lead to a significant relief oftumor burden in vivo, especially in combination with anti-PD-1 therapy.These findings may have important implications for harnessing chromatinand epigenetic regulators for onco-immunotherapy. In some embodiments,the immunotherapy is an antibody therapy (e.g., a monoclonal antibody, aconjugated antibody).

Provided herein are methods of treating cancer in a patient thatinclude: administering to a patient in need of cancer treatmenttherapeutically effective amounts of a lysine-specific demethylase 1A(LSD1) inhibitor and at least one of a programmed-cell death 1 (PD-1)inhibitor and a programmed-cell death ligand 1 (PD-L1) inhibitor, tothereby treat cancer in the patient.

Also provided herein are methods of treating cancer in a patient thatinclude: administering to a patient in need of cancer treatmenttherapeutically effective amounts of a lysine-specific demethylase 1A(LSD1) inhibitor and at least one immunotherapy, to thereby treat cancerin the patient.

In some embodiments of any of the methods described herein, the methodfurther includes identifying the patient as having cancer prior toadministering.

In some embodiments, the method includes administering a LSD1 inhibitorand a PD-1 inhibitor.

In some embodiments, the method includes administering a LSD1 inhibitor,a PD-1 inhibitor, and a PD-L1 inhibitor.

In some embodiments, the method includes administering a LSD1 inhibitorand a PD-L1 inhibitor.

In some embodiments, the at least one immunotherapy is selected from thegroup consisting of: an antibody, an adoptive cellular therapy, anantibody-drug conjugate, a toxin, a cytokine therapy, a cancer vaccine,a checkpoint inhibitor. In some embodiments, the method includes thecheckpoint inhibitor is a CTLA-4 inhibitor, a PD-1 inhibitor, a PD-L1inhibitor, a PD-L2 inhibitor, an OX40 (TNFRSF4) inhibitor, a TIM3 (TCell Immunoglobulin Mucin 3) inhibitor, or a LAG3 (Lymphocyte Activating3) inhibitor. In some embodiments, the PD-1 inhibitors blocks theinteraction of PD-1 with its ligands (e.g., PD-L1 or PD-L1).

In some embodiments of any of the methods described herein, the LSD1inhibitor is selected from the group consisting of: a small molecule, anantibody, and an inhibitory nucleic acid. In some embodiments whereinthe LSD1 inhibitor is an inhibitory nucleic acid, the inhibitory nucleicacid is a small interfering RNA or a short hairpin RNA. In someembodiments wherein the inhibitory nucleic acid is a short hairpin RNA,the short hairpin RNA includes SEQ ID NO: 2.

In some embodiments of any of the methods described herein, the LSD1inhibitor is a small molecule selected from the group consisting of:tranylcypromine, RN 1 dihydrochloride, GSK-LSD1, GSK2879552, ORY1001,GSK690, namoline, Cpd 2d, 52101, OG-L002, SP2509, CBB2007 and IMG-7289.

In some embodiments of any of the methods described herein, the PD-1inhibitor is selected from the group consisting of: a small molecule, anantibody, and an inhibitory nucleic acid.

In some embodiments wherein the PD-1 inhibitor is an inhibitory nucleicacid, the inhibitory nucleic acid is a small interfering RNA or a shorthairpin RNA. In some embodiments wherein the inhibitory nucleic acid isa short hairpin RNA, the short hairpin RNA includes e.g., SEQ ID NO: 4.

In some embodiments wherein the PD-1 inhibitor is an antibody, theantibody is nivolumab or pembrolizumab.

In some embodiments of any of the methods described herein, the PD-L1inhibitor is selected from the group consisting of: a small molecule, anantibody, and an inhibitory nucleic acid.

In some embodiments wherein the PD-L1 inhibitor is an inhibitory nucleicacid, the inhibitory nucleic acid is a small interfering RNA or a shorthairpin RNA. In some embodiments wherein the inhibitory nucleic acid isa short hairpin RNA, the short hairpin RNA includes e.g., SEQ ID NO: 6.

In some embodiments of any of the methods described herein, the PD-L1inhibitor is an antibody selected from the group consisting of:durvalumab, atezolizumab and avelumab.

In some embodiments of any of the methods described herein, the canceris a primary tumor.

In some embodiments of any of the methods described herein, the canceris a metastatic tumor.

In some embodiments of any of the methods described herein, the canceris selected from the group consisting of: melanoma, acute myeloidleukemia (AML), squamous cell carcinoma, renal cell carcinoma, non-smallcell lung cancer (NSCLC), small cell lung cancer (SCLC), gastric cancer,bladder cancer, kidney cancer, head and neck cancer, Ewing sarcoma,Hodgkin's lymphoma, Merkel cell carcinoma, breast cancer and prostatecancer.

In some embodiments of any of the methods described herein, the canceris a non-T-cell-infiltrating cancer.

In some embodiments of any of the methods described herein, the canceris a PD-1 and/or PD-L1 refractory cancer.

In some embodiments of any of the methods described herein, the canceris a PD-1 and/or PD-L1 resistant cancer.

In some embodiments of any of the methods described herein, the patienthas previously received cancer treatment.

In some embodiments of any of the methods described herein,administering occurs at least once a week.

In some embodiments of any of the methods described herein,administering is via intravenous, subcutaneous, intraperitoneal, rectal,and/or oral administration.

In some embodiments of any of the methods described herein, the LSD1inhibitor and the at least one PD-1 inhibitor or PD-L1 inhibitor areadministered simultaneously to the patient.

In some embodiments of any of the methods described herein, the LSD1inhibitor is administered to the patient prior to administration of thePD-1 inhibitor or PD-L1 inhibitor.

In some embodiments of any of the methods described herein, theadministration of the LSD1 inhibitor is stopped before theadministration of the PD-1 inhibitor or the PD-L1 inhibitor.

In some embodiments of any of the methods described herein, the methodfurther includes administering a chemotherapeutic agent.

In some embodiments of any of the methods described herein, treatingincludes reducing the volume of primary tumor in the patient.

In some embodiments of any of the methods described herein, treatingincludes delaying cancer progression in the patient.

In some embodiments of any of the methods described herein, treatingincludes modifying the tumor microenvironment of a cancer in thepatient.

In some embodiments of any of the methods described herein, treatingincludes sensitizing a cancer to a checkpoint inhibitor therapy.

In some embodiments of any of the methods described herein, treatingincludes decreasing the risk of developing at least one metastatic tumorin the patient.

In some embodiments of any of the methods described herein, treatingincludes decreasing the rate of and/or delaying tumor growth at ametastatic site.

In some embodiments of any of the methods described herein, treatingincludes decreasing tumor cell migration.

In some embodiments of any of the methods described herein, treatingincludes decreasing tumor cell invasion.

In some embodiments of any of the methods described herein, treatingincludes decreasing the rate of tumor growth in the patient.

In some embodiments of any of the methods described herein, treatingincludes eliciting tumor-intrinsic double-stranded RNA stress in acancer cell in the patient.

The present specification also provides compositions that are useful inthe methods described herein, e.g., combined compositions that include alysine-specific demethylase 1A (LSD1) inhibitor and at least oneimmunotherapy, e.g., at least one programmed-cell death 1 (PD-1)inhibitor and/or at least one programmed-cell death ligand 1 (PD-L1)inhibitor.

Also provided herein are methods of treating cancer in a patient thatinclude: administering to a patient in need of cancer treatmenttherapeutically effective amounts of a lysine-specific demethylase 1A(LSD1) inhibitor and at least one immunotherapy, to thereby treat cancerin the patient.

The term “treat” or “treatment” is used herein to denote delaying theonset of, inhibiting, alleviating the effects of, or prolonging the lifeof a patient suffering from, a condition, e.g., cancer. The terms“effective amount” and “amount effective to treat,” as used herein,refer to an amount or concentration of a composition or treatmentdescribed herein, e.g., an LSD1 inhibitor, utilized for a period of time(including acute or chronic administration and periodic or continuousadministration) that is effective within the context of itsadministration for causing an intended effect or physiological outcome.For example, effective amounts of a LSD1 inhibitor and an immunotherapy(e.g., any immunotherapy described herein) for use in the presentdisclosure include, for example, amounts that inhibit the growth ofcancer, e.g., tumors and/or tumor cells, improve delay tumor growth,improve survival for a patient suffering from or at risk for cancer, andimprove the outcome of other cancer treatments. As another example,effective amounts of a LSD1 inhibitor and an immunotherapy (e.g., anyimmunotherapy described herein) can include amounts that advantageouslyaffect a tumor microenvironment.

The term “patient” or “subject” is used throughout the specification todescribe an animal, human or non-human, to whom treatment according tothe methods of the present disclosure is provided. Veterinaryapplications are clearly anticipated by the present disclosure. The termincludes but is not limited to birds, reptiles, amphibians, and mammals,e.g., humans, other primates, pigs, rodents such as mice and rats,rabbits, guinea pigs, hamsters, cows, horses, cats, dogs, sheep andgoats. Preferred subjects are humans, farm animals, and domestic petssuch as cats and dogs.

Compositions and treatments described herein can be used to treatcellular proliferative and/or differentiation disorders. Examples ofcellular proliferative and/or differentiation disorders include cancer,e.g., carcinoma, sarcoma, metastatic disorders and hematopoieticneoplastic disorders, e.g., leukemias.

The term “cancer” refers to cells having the capacity for autonomousgrowth. Examples of such cells include cells having an abnormal state orcondition characterized by rapidly proliferating cell growth. The termis meant to include cancerous growths, e.g., tumors; oncogenicprocesses, metastatic tissues, and malignantly transformed cells,tissues, or organs, irrespective of histopathologic type or stage ofinvasiveness. Also included are malignancies of the various organsystems, such as respiratory, cardiovascular, renal, reproductive,hematological, neurological, hepatic, gastrointestinal, and endocrinesystems; as well as adenocarcinomas, which include malignancies such asmost colon cancers, renal-cell carcinoma, prostate cancer and/ortesticular tumors, non-small cell carcinoma of the lung, cancer of thesmall intestine, and cancer of the esophagus. Cancer that is “naturallyarising” includes any cancer that is not experimentally induced byimplantation of cancer cells into a subject, and includes, for example,spontaneously arising cancer, cancer caused by exposure of a patient toa carcinogen(s), cancer resulting from insertion of a transgeniconcogene or knockout of a tumor suppressor gene, and cancer caused byinfections, e.g., viral infections. The term “carcinoma” is artrecognized and refers to malignancies of epithelial or endocrinetissues. The term also includes carcinosarcomas, which include malignanttumors composed of carcinomatous and sarcomatous tissues. An“adenocarcinoma” refers to a carcinoma derived from glandular tissue orin which the tumor cells form recognizable glandular structures.

The term “sarcoma” is art recognized and refers to malignant tumors ofmesenchymal derivation. The term “hematopoietic neoplastic disorders”includes diseases involving hyperplastic/neoplastic cells ofhematopoietic origin. A hematopoietic neoplastic disorder can arise frommyeloid, lymphoid or erythroid lineages, or precursor cells thereof.

A metastatic tumor can arise from a multitude of primary tumor types,including but not limited to those of prostate, colon, lung, breast,bone, and liver origin. Metastases develop, e.g., when tumor cells shedfrom a primary tumor adhere to vascular endothelium, penetrate intosurrounding tissues, and grow to form independent tumors at sitesseparate from a primary tumor.

The term “PD-1 or PD-L1 refractory cancer” refers to a cancercharacterized by resistance to PD-1 inhibitor or PD-L1 inhibitortreatment. In some embodiments, the cancer is characterized by apopulation of cells (e.g., cancer cells or immune cells such as T cells)that have a reduced level of PD-1 or PD-L1 on the surface, or a reducedexpression of PD-1 or PD-L1 (e.g., as compared to non-cancer cells, ascompared to cells obtained from subjects without PD-1 or PD-L1refractory cancer, or as compared to a reference level or value), and/ora genetic lesion in a PD-1 or PD-L1 gene. The terms “a reduced level” or“a decreased level” is a reduction or decrease of PD-1 or PD-L1 of atleast a 1% (e.g., at least 2%, at least 4%, at least 6%, at least 8%, atleast 10%, at least 12%, at least 14%, at least 16%, at least 18%, atleast 20%, at least 22%, at least 24%, at least 26%, at least 30%, atleast 35%, at least 40%, at least 45%, at least 50%, at least 55%, atleast 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%, or at least 99%) reduction ascompared to a reference level or value.

The term “non-T-cell-infiltrating tumor” means a tumor that lacks Tcells within its tumor microenvironment. In some embodiments, anon-T-cell-infiltrating tumor is characterized by a population of cancercells that have down-regulated genes associated with T cell recognition,a reduced expression of polypeptides associated with T cell recognitionon its cell surface (e.g., a T-cell receptor), and/or T celldysfunction.

The term “population” when used before a noun means two or more of thespecific noun. For example, the phrase “a population of cancer cells”means “two or more cancer cells.” Non-limiting examples of cancer cellsare described herein.

A “chemotherapeutic agent” refers to a chemical compound useful in thetreatment of a cancer. Chemotherapeutic agents include, e.g.,“anti-hormonal agents” or “endocrine therapeutics” which act toregulate, reduce, block, or inhibit the effects of hormones that canpromote the growth of cancer. Additional classes, subclasses, andexamples of chemotherapeutic agents are known in the art.

Individuals considered at risk for developing cancer may benefit fromthe present disclosure, e.g., because prophylactic treatment can beginbefore there is any evidence and/or diagnosis of the disorder.Individuals “at risk” include, e.g., individuals exposed to carcinogens,e.g., by consumption (e.g., by inhalation and/or ingestion), at levelsthat have been shown statistically to promote cancer in susceptibleindividuals. Also included are individuals at risk due to exposure toultraviolet radiation, or their environment, occupation, and/orheredity, as well as those who show signs of a precancerous conditionsuch as polyps. Similarly, individuals in very early stages of cancer ordevelopment of metastases (i.e., only one or a few aberrant cells arepresent in the individual's body or at a particular site in anindividual's tissue) may benefit from such prophylactic treatment.

Skilled practitioners will appreciate that a patient can be diagnosed,e.g., by a medical professional, e.g., a physician or nurse (orveterinarian, as appropriate for the patient being diagnosed), assuffering from or at risk for a condition described herein, e.g.,cancer, using any method known in the art, e.g., by assessing apatient's medical history, performing diagnostic tests, and/or byemploying imaging techniques.

Skilled practitioners will also appreciate that treatment need not beadministered to a patient by the same individual who diagnosed thepatient (or the same individual who prescribed the treatment for thepatient). Treatment can be administered (and/or administration can besupervised), e.g., by the diagnosing and/or prescribing individual,and/or any other individual, including the patient her/himself (e.g.,where the patient is capable of self-administration).

Also contemplated by the present disclosure is administration of a LSD1inhibitor and an immunotherapy (e.g., any immunotherapy describedherein) to a patient in conjunction with at least one other treatment,e.g., chemotherapy, radiation therapy, gene therapy, and/or surgery, totreat conditions and disorders described herein (e.g., cancer).Alternatively or in addition, treatments described herein can beadministered in combination with chemotherapy. Chemotherapy can involveadministration of any of the following classes of compounds: alkylatingagents, antimetabolites, e.g., folate antagonists, purine antagonistsand/or pyrimidine antagonists; spindle poisons, e.g., vincas (e.g.,paclitaxel) and podophillotoxins; antibiotics, e.g., doxorubicin,bleomycin and/or mitomycin; nitrosoureas; inorganic ions, e.g.,cisplatin; biologic response modifiers, e.g., tumor necrosis factor-α(TNF-α) and interferon; enzymes, e.g., asparaginase; protein toxinsconjugated to targeting moieties; antisense molecules; and hormones,e.g, tomoxifen, leuprolide, flutamide, and megestrol. Alternatively orin addition, treatments described herein can be administered incombination with radiation therapy, e.g., using γ-radiation, neutronbeams, electron beams, and/or radioactive isotopes. Alternatively or inaddition, treatments described herein can be administered to patients incombination with immunotherapies other than administering a PD-1inhibitor, a PD-L1 inhibitor or a CTLA-4 inhibitor, e.g., administeringspecific effector cells, tumor antigens, and/or antitumor antibodies.Alternatively or in addition, treatments described herein can beadministered to patients in combination with gene therapy, e.g., theadministration of DNA encoding tumor antigens and/or cytokines. Methodsfor treating cancer, e.g., surgery, chemotherapy, immunotherapy, andradiotherapy, are more fully described in The Merck Manual of Diagnosisand Therapy, 17^(th) Edition, Section 11, Chapters 143 and 144, thecontents of which are expressly incorporated herein by reference intheir entirety.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this disclosure belongs. Methods and materials aredescribed herein for use in the present disclosure; other, suitablemethods and materials known in the art can also be used. The materials,methods, and examples are illustrative only and not intended to belimiting. All publications, patent applications, patents, sequences,database entries, and other references mentioned herein are incorporatedby reference in their entirety. In case of conflict, the presentspecification, including definitions, will control.

Other features and advantages of the disclosure will be apparent fromthe following detailed description and figures, and from the claims.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1A is a bar graph showing quantitative reverse transcriptionpolymerase chain reaction (RT-qPCR) analysis of selected endogenousretroviruses (ERVs) (HERV-E, HERV-F, HERV-K, HML-2, and ERVL), IFNs(IFN-α, IFN-β and IL-28) and ISGs (ISG15 and OASL) in human MCF-7 breastcancer cells treated with or without GSK-LSD1 for 6 days. The RT-qPCRdata were normalized to GAPDH and then relative to DMSO. RT-qPCR wasperformed in duplicates and repeated two to three times. Error barsrepresent the standard error of mean (SEM). *p<0.05, **p<0.01,***p<0.001, ns, not significant, as determined by unpaired t-test.

FIG. 1B is a picture of immunoblots showing shRNA-mediated knockdown ofLSD1 (sh-LSD1) in MCF-7 cells. Actin was used as a control for proteinlevel.

FIG. 1C is a bar graph showing shRNA-mediated knockdown of LSD1 in MCF-7cells (sh-LSD1) and shRNA against scramble (sh-Ctrl) in MCF-7 cells byRT-qPCR. The RT-qPCR data were normalized to GAPDH and then relative tosh-Ctrl. Error bars represent SEM from three experiments. *p<0.05,**p<0.01, ***p<0.001, ns, not significant, as determined by unpairedt-test.

FIG. 1D is a bar graph showing IFN-β secretion (pg/mL) in LSD1 knockdown(KD) MCF-7 cells detected by ELISA (n=3). Error bars represent standarddeviation (SD) between triplicates in one of two experiments. n.d., notdetected.

FIG. 1E is a picture of immunoblots showing LSD1 KD MCF-7 cells thatwere transduced with either wild type (WT) LSD1 or catalyticallyinactive LSD1 that harbors a K661A mutation (LSD1-K661A). Actin was usedas a control for protein level.

FIG. 1F is a bar graph showing RT-qPCR analysis of selected ERVs(HERV-E, HERV-F, HERV-K, HML-2, and ERVL) in MCF-7 cells transduced withshRNA against scramble (sh-C) or LSD1 (sh-LSD1). RT-qPCR was performedin duplicates and repeated two to three times. Error bars represent SEMfrom two experiments. *p<0.05, **p<0.01, ns, not significant, asdetermined by unpaired t-test.

FIG. 1G is a bar graph showing RT-qPCR analysis of IFN-α and IFN-β inMCF-7 cells transduced with shRNA against scramble (sh-C) or LSD1(sh-LSD1). RT-qPCR was performed in duplicates and repeated two to threetimes. Error bars represent the SEM from three experiments. *p<0.05,**p<0.01, ns, not significant, as determined by unpaired t-test.

FIG. 1H is a picture of immunoblots showing the protein expression ofDNMT proteins in MCF-7 cells with control shRNA or LSD1 KD.

FIG. 1I is bar graph showing 5-methyalcytosine content in genomic DNA ofcontrol and LSD1 KD MCF-7 cells as determined by HPLC-MS analysis.

FIG. 1J is a picture of immunoblots showing the protein expression ofISG15 in MCF-7 cells with control shRNA, LSD1 KD, or LSD1 KD rescuedwith LSD1.

FIG. 1K is a bar graph showing RT-qPCR analysis of selected ERVs(HERV-E, HERV-K, HML-2, and ERVL) and IFNs (IFN-β and IL-28) in humanT47D breast cancer cells transduced with shRNA against scramble or LSD1.The RT-qPCR data were normalized to GAPDH and then relative to sh-Ctrl.Error bars represent the standard deviation between triplicates.*p<0.05, **p<0.01, ***p<0.001, ns, not significant, as determined byunpaired t-test.

FIG. 1L is a bar graph showing RT-qPCR analysis of selected ERVs(HERV-E, HERV-K, HML-2, and ERVL) and IFNs (IFN-β and IL-28) in humanembryonic 293T kidney cells transduced with shRNA against scramble orLSD1. The RT-qPCR data were normalized to GAPDH and then relative tosh-Ctrl. Error bars represent the standard deviation between duplicates.*p<0.05, **p<0.01, ***p<0.001, ns, not significant, as determined byunpaired t-test.

FIG. 1M is volcano and M-A plots showing differentially expressed genesin LSD1 KD versus control MCF-7 cells as determined by RNA-seq. Dots ingrey represent significantly increased or decreased genes (FDR<0.05).

FIG. 1N is a representative dotmap showing the top 10 terms of a geneontology (GO) analysis of upregulated genes (log 2(FC)>1 and FDR <0.05)in LSD1 KD versus control MCF-7 cells. Dot size represents odds ratio.

FIG. 1O is representative dotmap showing the top 10 terms of a geneontology (GO) analysis of downregulated genes (log 2(FC)<−1 and FDR<0.05) in LSD1 KD versus control MCF-7 cells. Dot size represents oddsratio.

FIG. 2A is a Gene Set Enrichment Analysis (GSEA) analysis for responseto type 1 interferon (IFN) and antiviral response pathway in LSD1 KDversus WT control MCF-7 cells.

FIG. 2B is a plot showing LSD1 and H3K4me2 ChIP-seq signals at promoterregions of 125 induced interferon/antiviral responsive genes (IFN-gene,log 2(FC)>0 and FDR <0.05) or 537 selected genes with LSD1 peaks aspositive control (Pos-gene) in control (sh-Ctrl) and LSD1 KD (sh-LSD1)cells.

FIG. 2C is IGV images of TLR3 loci showing LSD1 and H3K4me2 levels inLSD1 KD and control MCF-7 cells.

FIG. 2D are IGV images of SED1A loci showing LSD1 and H3K4me2 levels inLSD1 KD and control MCF-7 cells.

FIG. 2E is a heatmap for differential transcript expression ofrepetitive elements between LSD1 KD and WT control.

FIG. 2F is heatmaps showing differential expression of sense orantisense transcripts of ERVs between LSD1 KD and WT control.

FIG. 2G is plots showing LSD1 and H3K4me2 ChIP-seq signals at genomicloci of 8593 individual ERVs from 279 ERV subfamilies in control andLSD1 KD cells.

FIG. 2H is plots of LSD1 and H3K4me2 ChIP-seq signals at genomic loc ofHERV-E subfamily in control and LSD1 KD cells.

FIG. 2I is histogram plots of normalized ChIP-Seq tag intensities ofLSD1 and H3K4me2 at HERV-E loci of 7813 bp in length.

FIG. 2J is a bar graph showing fold changes of reverse complementarysense-/antisense transcripts (overlapping) and extra sense or antisensetranscripts (extra) of a number of retrotransposons between LSD1 KD andcontrol cells determined by directional RNA-Seq

FIG. 2K is a picture of a PCR gel showing PCR amplification of selectedERVs using strand specific primers in MCF-7 cells with sh-C or sh-LSD1.An asterisk indicates non-specific bands.

FIG. 2L is a bar graph showing RT-qPCR analysis of EGFP, engineeredHERV-(K+E) in MCF-7 cells transduced with pHAGE-EGFP orpHAGE-HERV-(K+E). The RT-qPCR data were normalized to GAPDH and thenrelative to untransduced cells. Error bars represent SEM from threeexperiments two experiments. *p<0.05, **p<0.01, ***p<0.001, ns, notsignificant, as determined by unpaired t-test.

FIG. 2M is a bar graph showing RT-qPCR analysis of IFNα, IFNβ, IL-28,ISG15 and OASL in MCF-7 cells transduced with pHAGE-EGFP orpHAGE-HERV-(K+E). The RT-qPCR data were normalized to GAPDH and thenrelative to untransduced cells. Error bars represent SEM from threeexperiments two experiments. *p<0.05, **p<0.01, ***p<0.001, ns, notsignificant, as determined by unpaired t-test.

FIG. 2N is a bar graph showing double-stranded RNA (dsRNA) enrichment ofselected retrotransposons (HERV-E, HERV-F, HERV-K, ERVL, Syn-1, Line1and AluYA5) in control (sh-C) and LSD1 KD (sh-LSD1) MCF-7 cells byRT-qPCR. Total RNA extract from control or LSD1 KD MCF-7 cells wasdigested with RNase A versus mock under high salt condition (350 mMNaCl), followed by a second round of RNA extraction with TRIzol. Theratios of (retrotransposon/GAPDH)RNase/(retrotransposon/GAPDH)mock werecalculated as enrichment fold. GAPDH was used as an internal control.Error bars represent SEM from three experiments. p<0.05, **p<0.01,***p<0.001, ns, not significant, as determined by unpaired t-test.

FIG. 2O is a bar graph showing RT-qPCR analysis of selectedretrotransposon transcripts (HERV-E, HERV-F, HERV-K, ERVL, Syn-1, Line1,AluYA5) and GAPDH captured by a dsRNA-specific antibody (J2) pulldownassay in MCF-7 cells with sh-C or sh-LSD1. Error bars represent SDbetween duplicates. *p<0.05, **p<0.01, ***p<0.001, ns, not significant,as determined by unpaired t-test.

FIG. 2P is a heatmap showing the expression nucleic acid receptors incontrol and LSD1 KD MCF-7 cells as determined by RNA-seq.

FIG. 2Q is a representative immunoblot of TLR3, MDA5 and RIG-I incontrol and LSD1 KD cells.

FIG. 3A is a picture of immunoblots showing TLR3, MDA5 and RIG-Iexpression in control (sh-C), LSD1 KD (sh-LSD1), LSD1/TLR3 DKO(sh-LSD1+sh-TLR3), LSD1/MDA5 DKO (sh-LSD1+sh-MDA5), or LSD1/RIG-I DKO(sh-LSD1+sh-RIG-I) MCF-7 cells.

FIG. 3B is a bar graph showing RT-qpCR analysis of TLR3, selected ERVs(HERV-E, HERV-K and HML-2), IFNs (IFN-β and IL-28) and ISGs (ISG15 andOASL) in MCF-7 cells transduced with shRNA against scramble, LSD1 orLSD1 and TLR3. RT-qPCR was performed in duplicates and repeated two tothree times. Error bars represent standard deviation (SD). p<0.05,**p<0.01, ***p<0.001, ns, not significant as determined by unpairedt-test.

FIG. 3C is a bar graph showing RT-qPCR analysis of MDA5, selected ERVs(HERV-E, HERV-K and HML-2), IFNs (IFN-β and IL-28) and ISGs (ISG15 andOASL) in MCF-7 cells transduced with shRNA against scramble, LSD1 orLSD1 and MDA5. RT-qPCR was performed in duplicates and repeated two tothree times. Error bars represent standard deviation (SD). *p<0.05,**p<0.01, ***p<0.001, ns, not significant as determined by unpairedt-test.

FIG. 3D is a bar graph showing RT-qPCR analysis of RIG-I, selected ERVs(HERV-E, HERV-K and HML-2), IFNs (IFN-β and IL-28) and ISGs (ISG15 andOASL) in MCF-7 cells transduced with shRNA against scramble, LSD1 orLSD1 and RIG-I. RT-qPCR was performed in duplicates and repeated two tothree times. Error bars represent standard deviation (SD). *p<0.05,**p<0.01, ***p<0.001, ns, not significant as determined by unpairedt-test.

FIG. 3E is a bar graph showing RT-qPCR analysis of MAVS, selected ERVs(HERV-E, HERV-K and HML-2), IFNs (IFN-β and IL-28) and ISGs (ISG15 andOASL) in MCF-7 cells transduced with shRNA against scramble, LSD1 orLSD1 and MAVS. Error bars represent standard deviation betweenduplicates. *p<0.05, **p<0.01, ***p<0.001, ns, not significant asdetermined by unpaired t-test.

FIG. 3F is a picture of immunoblots showing cGAS and STING proteins inMCF-7+sh-Ctrl, sh-LSD1, sh-LSD1+shcGAS and sh-LSD1+shSTING cells.

FIG. 3G is a bar graph showing RT-qPCR analysis of HERV-E, HERV-K,HML-2, IL-28, ISG15 and OASL in MCF-7+sh-Ctrl cells, MCF-7+sh-LSD1cells, MCF-7+sh-LSD1+sh-cGAS cells, and MCF-7+sh-LSD1+sh-STING cells.The RT-qPCR data were normalized to GAPDH and then relative to sh-Ctrl.Error bars represent SEM from three experiments. *p<0.05, **p<0.01,***p<0.001, ns, not significant, as determined by unpaired t-test.

FIG. 3H is a bar graph showing RT-qPCR analysis of IFN-β, IL-28, OASLand ISG15 in control, cGAS KD and STING KD MCF-7 transfected withfragmented genomic DNA from mammalian cells or mock transfected. TheRT-qPCR data were normalized to GAPDH and then relative to sh-Ctrl.Error bars represent SD between duplicates in one of two experiments.*p<0.05, **p<0.01, ***p<0.001, ns, not significant, as determined byunpaired t-test.

FIG. 4A is a picture of immunoblots showing shRNA-mediated knockdown ofLSD1 in MCF-7 cells (sh-LSD1), rescue with WT LSD1 or catalyticallyinactive LSD1-K661A. Actin was used as a control for protein level. Theprotein expression of core components (DICER, AGO2 and TRBP2) of theRISC complex was measured by immunoblot.

FIG. 4B is a picture of immunoblots showing protein expression of LSD1and Drosha in MCF-7 cells transduced with control shRNA (sh-C) or LSD1shRNA (sh-LSD1). Actin was used as a control for protein level.

FIG. 4C is a picture of immunoblots showing GFP and GFPL proteinexpression in U2OS cells expressing dual reporters GFPL/GFP-let-7 andtransduced with shRNA against scramble, LSD1 or AGO2. Actin was used asa control for protein level.

FIG. 4D is a picture of immunoblots showing ISG15 protein expression inMCF-7+sh-C cells, MCF-7+sh-LSD1 cells, MCF-7+sh-LSD1+sh-TLR3 cells,MCF-7+sh-LSD1+sh-RIG-I cells, and MCF-7+sh-LSD1+sh-MDA5 cells. Actin wasused as a control for protein level.

FIG. 4E is a picture of immunoblots showing MAVS protein expression inMCF-7+sh-C cells, MCF-7+sh-LSD1 cells, MCF-7+sh-LSD1+sh1-MAVS cells, andMCF-7+sh-LSD1+sh2-MAVS cells. Actin was used as a control for proteinlevel.

FIG. 4F is a bar graph showing relative let-7 miRISC activity byquantifying GFP and GFPL protein signals in U2OS cells expressing dualreporters GFPL/GFP-let-7 and transduced with shRNA against scramble,LSD1 or AGO2. Error bars represent SD between duplicates. p<0.05,**p<0.01, ***p<0.001, ns, not significant, as determined by unpairedt-test.

FIG. 4G is a bar graph showing the ratios of GFPL over GFP protein indifferent samples from five repeats for sh-LSD1 and two repeats forsh-AGO2. The ratio in control shRNA sample was considered as 100% miRISCactivity.

FIG. 4H is a bar graph showing double-stranded RNA (dsRNA) enrichment ofselected retrotransposons (HERV-E, HERV-F, HERV-K, ERVL, Syn-1, Line1and AluYA5) in control (sh-C) and AGO2 KD (sh-AGO2) MCF-7 cells byRT-qPCR. Total RNA extract from control or LSD1 KD MCF-7 cells wasdigested with RNase A versus mock under high salt condition (350 mMNaCl), followed by a second round of RNA extraction with TRIzol. Theratios of (retrotransposon/GAPDH)RNase/(retrotransposon/GAPDH)mock werecalculated as enrichment fold. GAPDH was used as an internal control.RT-qPCR was performed in duplicates and repeated two to three times.Error bars represent SD between duplicates. p<0.05, **p<0.01,***p<0.001, ns, not significant, as determined by unpaired t-test.

FIG. 4I is a bar graph showing RT-qPCR analysis of selected IFNs (IFN-βand IL-28), ISGs (OASL and ISG15), TLR3, MDA5 and RIG-I in MCF-7 cellstransduced with shRNA against scramble or AGO2. RT-qPCR was performed induplicates and repeated twice. Error bars represent standard deviation.p<0.05, **p<0.01, ***p<0.001, ns, not significant, as determined byunpaired t-test.

FIG. 4J is a picture of immunoblots showing protein expression of MDA5,RIG-I and ISG15 in the same cells used in FIG. 4I. Actin was used as acontrol for protein level.

FIG. 4K is bar graph showing RT-qPCR analysis of IFN-β, IL-28, ISG15,OASL, TLR3, MDA5 and RIG-I in MCF-7 cells transduced with shRNA againstscramble (sh-Ctrl) or DICER (sh4-DICER). The RT-qPCR data werenormalized to GAPDH and then relative to sh-Ctrl. Error bars representSD between duplicates in one experiment. *p<0.05, **p<0.01, ***p<0.001,ns, not significant, as determined by unpaired t-test.

FIG. 4L is a picture of immunoblots showing protein expression of DICERin MCF-7+sh-Ctrl, MCF-7+sh1-DICER, MCF-7+sh4-DICER cells. Actin was usedas a control for protein level.

FIG. 4M is bar graph showing RT-qPCR analysis of IFN-β, IL-28, ISG15,OASL, TLR3, MDA5 and RIG-I in MCF-7 cells transduced with shRNA againstscramble (sh-Ctrl) or TRBP2 (sh4-TRBP2). The RT-qPCR data werenormalized to GAPDH and then relative to sh-Ctrl. Error bars representSD between duplicates in one experiment. *p<0.05, **p<0.01, ***p<0.001,ns, not significant, as determined by unpaired t-test.

FIG. 4N is a picture of immunoblots showing protein expression of TRBP2in MCF-7+sh-Ctrl, MCF-7+sh1-TRBP2, MCF-7+sh4-TRBP2 cells. Actin was usedas a control for protein level.

FIG. 4O is a picture of immunoblots showing protein expression of AGO2and LSD1 in MCF-7+sh-LSD1 cells and MCF-7+FH-AGO2 cells. Actin was usedas a control for protein level.

FIG. 4P is a bar graph showing dsRNA enrichment of retrotransposons(HERV-E, HERV-F, HERV-K, ERVL, Line1 and AluYA5) in MCF-7+sh-controlcells, MCF-7+sh-LSD1 cells, MCF-7+FH-AGO2+sh-control cells, andMCF-7+FH-AGO2+sh-LSD1 cells. Error bars represent standard error of themean (SEM) from five experiments. *p<0.05, **p<0.01, ***p<0.001, ns, notsignificant, as determined by unpaired t-test.

FIG. 4Q is a bar graph showing RNA levels of HERV-E, HERV-K, HML-2,IFNβ, IL-28, ISG15 and OASL in MCF-7+sh-control cells, MCF-7+sh-LSD1cells, MCF-7+FH-AGO2+sh-control cells, and MCF-7+FH-AGO2+sh-LSD1 cells.Error bars represent SD between triplicates. p<0.05, **p<0.01,***p<0.001, ns, not significant, as determined by unpaired t-test.

FIG. 5A is a picture of immunoblots showing the protein expression ofcore components of the RISC complex (DICER, AGO2 and TRBP2) inMCF-7+sh-C cells, MCF-7+sh-LSD1 cells, MCF-7+sh-LSD1+LSD1 cells, andMCF-7+sh-LSD1+LSD1-K661A cells. Actin was used as a control for proteinlevel.

FIG. 5B is a bar graph showing RT qPCR analysis of AGO1-4, DICER andTRBP2 in control and LSD1 KD MCF-7 cells. Data was normalized to GAPDHand relative to sh-Ctrl. Error bars represent SEM from two experiments.*p<0.05, **p<0.01, ns, not significant, as determined by unpairedt-test.

FIG. 5C is a picture of immunoblots showing the protein expression ofAGO2 in MCF-7 cells treated with 50 μg/ml cycloheximide (CHX) in thepresence of absence of 2 μM GSK-LSD1 at 0, 3, 6, 9, 12, hours. Actin wasused as a control for protein level.

FIG. 5D is a graph showing the quantification of AGO2 signal from fiveexperiments of MCF-7 cells treated with 50 μg/ml cycloheximide (CHX) inthe presence of absence of 2 μM GSK-LSD1 at 0, 3, 6, 9, 12, hours. Errorbars represent SEM from five experiments. *p<0.05, **p<0.01, ns, notsignificant, as determined by unpaired t-test.

FIG. 5E is a picture of immunoblots showing the physical interactionbetween LSD1 and AGO2 by co-immunoprecipitation assay using whole celllysate (WCL) of MCF-7 cells stably expressing FH-AGO2.

FIG. 5F is a picture of immunoblots showing the physical interactionbetween LSD1 and TRBMP2 by co-immunoprecipitation assay using whole celllysate (WCL) of MCF-7 cells stably expressing FH-TRBP2.

FIG. 5G is a picture of immunoblots showing protein expression of DICER,AGO2, TRBP2 and LSD1 in whole cell lysate (WCL), cytoplasm (CytoE) andnuclear lysate (NE).

FIG. 5H is a picture of immunoblots showing the physical interactionbetween LSD1 and AGO2 by co-immunoprecipitation assay using nuclearlysate (NE) of MCF-7 cells stably expressing FH-LSD1.

FIG. 5I is a picture of immunoblots showing purified FH-AGO2 from MCF-7cells treated by LSD1 KD or GSK-LSD1 as determined by mass spectrometryfor the identification of lysine methylation. sh-Ctrl (VGKSGNIPAGTTVDTK;SEQ ID NO: 156) and sh-LSD1, GSK-LSD1(VGK(me)SGNIPAGTTVDTK; SEQ ID NO:157).

FIG. 5J is a dot plot detecting the reactivity of K726mel-specificantibody against un-, mono- or di-methylated AGO2 peptides.

FIG. 5K is a picture of immunoblots showing ectopically expressed wildtype FH-AGO2, FH-AGO2-K726R and FH-AGO2-K726A in MCF-7 cells treatedwith LSD1 KD co-immunoprecipitated by α-Flag and immunoblotted withmono-methyl AGO2 specific antibody.

FIG. 5L is a picture of immunoblots showing ectopically expressed wildtype FH-AGO2, FH-AGO2-K726R and FH-AGO2-K726A in MCF-7 cells treatedwith GSK-LSD1 co-immunoprecipitated by α-HA and immunoblotted withmono-methyl AGO2 specific antibody.

FIG. 5M is a picture of immunoblots showing K726mel on endogenous AGO2in control or LSD1 KD MCF-7 cells.

FIG. 5N is a picture of immunoblots showing AGO2 mono-methylation withimmunoprecipitated proteins from MCF-7 cells.

FIG. 5O is a bar graph showing signal intensities of AGO2mono-methylation status at K726 in in vitro demethylation assay withimmunoprecipitated proteins from MCF-7 cells. Error bars represent SDbetween duplicates in one experiment, or represent SEM from threeexperiments. *p<0.05, **p<0.01, ***p<0.001, ns, not significant, asdetermined by unpaired t-test.

FIG. 5P is a graph showing protein stability of transiently expressedwild type FH-AGO2 and FH-AGO2-K726R in 293T cells measured using CHXchase assay in the presence or absence of 2 μM GSK-LSD1. The averagedAGO2 quantification from two experiments was shown.

FIG. 6A is bar graph showing RT-qPCR analysis of selectedretrotransposons (MuSD, MuERV-L, Line1 and IAP), IFNs (IFN-α, IFN-β andIL-28) and ISG15, OASL, TLR3, MDA5 and RIG-I in murine B16 melanomacells transduced with gRNA against scramble (scramble) or LSD1 (LSD1 KO,clone g4-7) (n=2). Data was normalized to GAPDH relative to sh-Ctrl.Error bars represent SEM from two experiments. *p<0.05, **p<0.01,***p<0.001, ****p<0.0001, ns, not significant, as determined by unpairedt-test.

FIG. 6B is bar graph showing RT-qPCR analysis of MuERV-L, Line 1, IFN-βand ISG15 in murine Lewis lung carcinoma (LLC) cells transduced withshRNA against scramble (scramble), LSD1 KO1 and LSD1 K02. The RT-qPCRdata were normalized to GAPDH and then relative to scramble. Error barsrepresent SEM from three experiments. *p<0.05, **p<0.01, ***p<0.001, ns,not significant, as determined by unpaired t-test.

FIG. 6C is bar graph showing RT-qPCR analysis of MuERV-L, Line 1, IFN-α,IFN-β, IL-28, ISG15 and OASL in D4m cells transduced with shRNA againstscramble (scramble), LSD1 KO1 and LSD1 K02. The RT-qPCR data werenormalized to GAPDH and then relative to scramble. Error bars representSEM from three experiments. *p<0.05, **p<0.01, ***p<0.001, ns, notsignificant, as determined by unpaired t-test.

FIG. 6D is bar graph showing RT-qPCR analysis of MuERV-L, Line 1, IFN-α,IFN-β, IL-28, ISG15 and OASL in B16 cells transduced with shRNA againstscramble (scramble), LSD1 KO1 and LSD1 K02. The RT-qPCR data werenormalized to GAPDH and then relative to scramble. Error bars representSEM from three experiments.

FIG. 6E is a bar graph showing double-stranded RNA (dsRNA) enrichment ofselected retrotransposons (MuSD, MuERV-L, Line1 and IAP) in control orLSD1 KO B16 cells. Total RNA extract from control or LSD1 KD MCF-7 cellswas digested with RNase A versus mock under high salt condition (350 mMNaCl), followed by a second round of RNA extraction with TRIzol. Theratios of (retrotransposon/Actin)RNase/(retrotransposon/Actin)mock werecalculated as enrichment fold. Error bars represent SEM betweentriplicates. *p<0.05, **p<0.01, ***p<0.001, ns, not significant, asdetermined by unpaired t-test.

FIG. 6F is a picture of Hybond N+ membranes immunoblotted with adsRNA-specific antibody (J2) using total RNA extracted from scramble orLSD1 KO B16 cells and treated with mock, RNase Tl, RNase III or RNase A(350 mM NaCl), followed by a second round of RNA extraction with TRIzol.

FIG. 6G is a bar graph showing double-stranded RNA (dsRNA) enrichment ofMuERV-L, MuSD, IAP and Line1 in control and LSD1 KO D4m cells byRT-qPCR. Total RNA extract from control or LSD1 KO D4m cells wasdigested with RNase A versus mock under high salt condition (350 mMNaCl), followed by a second round of RNA extraction with TRIzol. Theratios of (retrotransposon/GAPDH)RNase/(retrotransposon/GAPDH)mock werecalculated as enrichment fold. GAPDH was used as an internal control.Error bars represent SEM from three experiments. p<0.05, **p<0.01,***p<0.001, ns, not significant, as determined by unpaired t-test.

FIG. 6H is a picture of a crystal violet cell proliferation assay ofB16+sh-Ctrl cells and B16+sh1-LSD1 cells, after 6 days of growth beforecrystal violet staining.

FIG. 6I is a bar graph showing the relative colony area of theproliferation assay of FIG. 6H relative to B16+sh-Ctrl. Error barsrepresent SD between triplicates in one of two experiments. *p<0.05,**p<0.01, ***p<0.001, ****p<0.0001, ns, not significant, as determinedby unpaired t-test.

FIG. 6J is a picture of a crystal violet cell proliferation assay of B16(LSD1 KO, clone g4-7), after 6 days of growth before crystal violetstaining.

FIG. 6K is a bar graph showing the relative colony area of theproliferation assay of FIG. 6J relative to scramble. Error barsrepresent SD between triplicates in one of two experiments. *p<0.05,**p<0.01, ***p<0.001, ****p<0.0001, ns, not significant, as determinedby unpaired t-test.

FIG. 6L is a representative image of sequencing results of genomic Lsd1,Mda5, Ifnar1, Ifnb and Tlr3 exons targeted by gRNAs in corresponding B16clones and in alignment with reference sequences. From top to bottom:B16 CRISPR-LSD1, clone gRNA4-A7 (SEQ ID NO: 122), reference (SEQ ID NO:123); B16 CRISPR-LSD1, clone gRNA5-4 (SEQ ID NO: 124), reference (SEQ IDNO: 125); B16 CRISPR-MDA5, clone gRNA4-16 (SEQ ID NO: 126), reference(SEQ ID NO: 127); B16 CRISPR-MDA5, clone gRN4-16 (SEQ ID NO: 128),reference (SEQ ID NO: 129); B16 CRISPR-LSD1/MDA5, clone gRNA4-19 (SEQ IDNO: 130), reference (SEQ ID NO: 131); B16 CRISPR-LSD1/MDA5, clonegRNA4-19 (SEQ ID NO: 132), reference (SEQ ID NO: 133); B16CRISPR-IFNAR1, clone gRNA1-10 (SEQ ID NO: 134), reference (SEQ ID NO:135); B16 CRISPR-IFNAR1, clone gRNA1-10 (SEQ ID NO: 136), reference (SEQID NO: 137); B16 CRISPR-LSD1/IFNAR1, clone gRNA1-16 (SEQ ID NO: 138),reference (SEQ ID NO: 139); B16 CRISPR-IFNβ, clone gRNA3-14 (SEQ ID NO:140), reference (SEQ ID NO: 141); B16 CRISPR-LSD1/IFNβ, clone gRNA3-16(SEQ ID NO: 142), reference (SEQ ID NO: 143); B16 CRISPR-LSD1/TLR3,clone gRNA6-7 (SEQ ID NO: 144), reference (SEQ ID NO: 145).

FIG. 6M is a picture of immunoblots showing LSD1 and MDA5 expression inCRISPR/Cas9-modified B16 cells (scramble, LSD1 KO, and LSD1/MDA5 DKO).

FIG. 6N is a picture of a crystal violet cell proliferation assay of B16scramble cells, B16 LSD1 KO cells and B16 LSD1/MDA5 KO cells, after 6days of growth before crystal violet staining.

FIG. 6O is a bar graph showing the relative colony area of theproliferation assay of FIG. 6N relative to B16 scramble. Error barsrepresent SD between quadruplicates in one of two experiments. *p<0.05,**p<0.01, ***p<0.001, ****p<0.0001, ns, not significant, as determinedby unpaired t-test.

FIG. 6P is a bar graph showing RT-qPCR analysis of selectedretrotransposons (MuERV-L and Line1) and IFNs (IFN-α, IFN-β and IL-28),OASL, ISG15, TLR3 and RIG-I in B16 scramble cells, B16 LSD1 KO cells andB16 LSD1/MDA5 KO cells. Error bars represent SEM between duplicates.*p<0.05, **p<0.01, ***p<0.001, ns, not significant, as determined byunpaired t-test.

FIG. 6Q is a picture of Hybond N+ membranes immunoblotted with adsRNA-specific antibody (J2) using total RNA extracted from scramble,LSD1 KO or LSD1/MDA5 DKO B16 cells and treated with mock, RNase Ti,RNase III or RNase A (350 mM NaCl), followed by a second round of RNAextraction with TRIzol.

FIG. 7A is a representative image of sequencing results of genomic Lsd1exon targeted by gRNA5 in corresponding LLC clones and in alignment withreference sequences. From top to bottom: LLC CRISPR-LSD1, clonegRNA5-A29 (SEQ ID NO: 146), reference (SEQ ID NO: 147); LLC CRISPR-LSD1,clone gRNA5-B30 (SEQ ID NO: 148), reference (SEQ ID NO: 149).

FIG. 7B is a picture of immunoblots of LSD1 in CRISPR/Cas9-modified LLCclones.

FIG. 7C is a representative image of sequencing results of genomic Lsd1exons targeted by two gRNAs in corresponding D4m clones and in alignmentwith reference sequences. From top to bottom: D4m CRISPR-LSD1, clonegRNA5-B37 (SEQ ID NO: 150), reference (SEQ ID NO: 151); D4m CRISPR-LSD1,clone gRNA3-8 (SEQ ID NO: 152), reference (SEQ ID NO: 153) and D4mCRISPR-LSD1, clone gRNA3-8 (SEQ ID NO: 154), reference (SEQ ID NO: 155).

FIG. 7D is a picture of immunoblots of LSD1 in CRISPR/Cas9-modified D4mclones.

FIG. 7E is a picture of immunoblots of LSD1 with two antibodies inCRISPR/Cas9-modified B16 clones transfected with different gRNAstargeting Lsd1.

FIG. 7F is a picture of immunoblots of LSD1 in CRISPR/Cas9-modified B16clones transfected with different gRNAs targeting Lsd1.

FIG. 8A is a bar graph showing RT-qPCR analysis of MuERV-L, Line1,IFN-α, IFN-β, IL-28, OASL and ISG15 in B16 scramble cells and MDA5 KOB16 cells. The RT-qPCR data were normalized to GAPDH and then relativeto scramble. Error bars represent SD between duplicates. *p<0.05,**p<0.01, ***p<0.001, ns, not significant, as determined by unpairedt-test.

FIG. 8B is a picture of a crystal violet cell proliferation assay of B16scramble cells, B16 LSD1 KO cells and B16 LSD1/MDA5 KO cells, after 6days of growth before crystal violet staining.

FIG. 8C is a bar graph showing the relative colony area of theproliferation assay of FIG. 8B relative to B16 scramble. Error barsrepresent SD between quadruplicates in one of two experiments. *p<0.05,**p<0.01, ***p<0.001, ****p<0.0001, ns, not significant, as determinedby unpaired t-test.

FIG. 8D is a bar graph showing RT-qPCR analysis of IFN-α, IFN-β, IL-28,OASL and ISG15 in B16 scramble cells and TLR3 KO B16 cells. The RT-qPCRdata were normalized to GAPDH and then relative to scramble. Error barsrepresent SD between duplicates. *p<0.05, **p<0.01, ***p<0.001, ns, notsignificant, as determined by unpaired t-test.

FIG. 8E is a bar graph showing RT-qPCR analysis of IFN-α, IFN-β, IL-28OASL and ISG15 in B16 scramble cells, B16 LSD1 KO cells and B16LSD1/IFNAR1 KO cells. Error bars represent SD between duplicates.*p<0.05, **p<0.01, ***p<0.001, ns, not significant, as determined byunpaired t-test.

FIG. 8F is a picture of a crystal violet cell proliferation assay of B16scramble cells, B16 LSD1 KO cells and B16 LSD1/IFNAR1 KO cells, after 6days of growth before crystal violet staining.

FIG. 8G is a bar graph showing the relative colony area of theproliferation assay of FIG. 8F relative to B16 scramble. Error barsrepresent SD between triplicates in one of two experiments. *p<0.05,**p<0.01, ***p<0.001, ****p<0.0001, ns, not significant, as determinedby unpaired t-test.

FIG. 8H is a picture of immunoblots showing IFNAR1 expression inCRISRP/Cas9-modified B16 cells as indicated.

FIG. 8I is a picture of a crystal violet cell proliferation assay of B16scramble cells, B16 LSD1 KO cells, B16 IFN-β KO cells and B16 LSD1/IFN-βKO cells, after 6 days of growth before crystal violet staining.

FIG. 8J is a bar graph showing the relative colony area of theproliferation assay of FIG. 8J relative to B16 scramble. Error barsrepresent SD between triplicates in one of two experiments. *p<0.05,**p<0.01, ***p<0.001, ****p<0.0001, ns, not significant, as determinedby unpaired t-test.

FIG. 8K is a bar graph showing RT-qPCR analysis of MuERV-L, Line1,IFN-α, IL-28, ISG15, OASL, TLR3, MDA5, RIG-I in B16 scramble cells andLSD1/IFN-βKO B16 cells. The RT-qPCR data were normalized to GAPDH andthen relative to scramble. Error bars represent SD between duplicates.*p<0.05, **p<0.01, *** p<0.001, ns, not significant, as determined byunpaired t-test.

FIG. 8L is bar graph showing mouse IFN-γ levels in scramble, IFN-β KO,LSD1/IFN-β DKO B16 cells challenged by poly(I:C), as determined byenzyme-linked immunosorbent assay (ELISA).

FIG. 9A is a line graph showing tumor growth of immunocompetent miceinoculated with 500k scramble (n=14) or LSD1 KO B16 cells (n=11). Errorbars represent SEM of individual mice in one experiment. Data representstwo independent experiments. *p<0.05, **p<0.01, ***p<0.001,****p<0.0001, ns, not significant, as determined by unpaired t-test.

FIG. 9B is a line graph showing survival of immunocompetent miceinoculated with 500k scramble or LSD1 KO B16 cells. Data represents twoindependent experiments. Error bars represent SEM of individual mice inone experiment. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, ns, notsignificant, as determined by log-rank test.

FIG. 9C is a line graph showing tumor growth of immunodeficient mice(TCRα KO) or immunocompetent mice inoculated with 500k scramble or LSD1KO B16 cells. Error bars represent SEM of individual mice in oneexperiment. Data represents two independent experiments. *p<0.05,**p<0.01, ***p<0.001, ****p<0.0001, ns, not significant, as determinedby ANOVA.

FIG. 9D is a line graph showing survival of immunodeficient mice (TCRαKO) or immunocompetent mice inoculated with 500k scramble or LSD1 KO B16cells. Data represents two independent experiments. Error bars representSEM of individual mice in one experiment. *p<0.05, **p<0.01, ***p<0.001,****p<0.0001, ns, not significant, as determined by log-rank test.

FIG. 9E is a line graph showing tumor growth of immunocompetent miceinoculated with 500k scramble B16 cells, LSD1 KO B16 cells, MDA5 KO B16cells, or LSD1/MDA5 DKO B16 cells. Error bars represent SEM ofindividual mice in one experiment. Data represents two independentexperiments. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, ns, notsignificant, as determined by ANOVA.

FIG. 9F is a line graph showing survival of immunocompetent miceinoculated with 500k scramble B16 cells, LSD1 KO B16 cells, MDA5 KO B16cells, or LSD1/MDA5 DKO B16 cells. Data represents two independentexperiments. Error bars represent SEM of individual mice in oneexperiment. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, ns, notsignificant, as determined by log-rank test.

FIG. 9G is a line graph showing tumor growth of immunocompetent miceinoculated with 500k scramble B16 cells, LSD1 KO B16 cells, IFN-β KO B16cells, or LSD1/IFN-β DKO B16 cells. Error bars represent SEM ofindividual mice in one experiment. Data represents two independentexperiments. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, ns, notsignificant, as determined by ANOVA.

FIG. 9H is representative images of lung metastasis in immunocompetentmice receiving 200k scramble or LSD1 KO B16 cells intravenously taken 14days post-injection.

FIG. 9I is a dot plot showing the quantification of lung metastasisimmunocompetent mice receiving 200k scramble or LSD1 KO B16 cellsintravenously.

FIG. 10A is bar graphs showing the number of tumor infiltratinglymphocytes (TILs) per gram of B16 tumor in immunocompetent mice (n=5for scramble, n=5 for LSD1 KO and n=6 for LSD1/MDA5 DKO) as determinedby flow cytometry at day 14 when tumor sizes were comparable among thetested groups. Data represents two independent experiments. Error barsrepresent SEM of individual mice in one experiment. *p<0.05, ***p<0.001,****p<0.0001, ns, not significant, as determined by unpaired t-test.

FIG. 10B is bar graphs showing T cells in draining lymph nodes (dNLs) ofB16 tumor-bearing immunocompetent mice (n=5 for scramble, n=5 for LSD1KO and n=6 for LSD1/MDA5 DKO).

FIG. 10C is bar graphs showing the percentage of granzyme B positive(GzmB+) or Ki-67+CD8+TILs as in 10A. Error bars represent SEM ofindividual mice in one experiment. *p<0.05, ***p<0.001, ****p<0.0001,ns, not significant, as determined by unpaired t-test.

FIG. 10D is bar graphs showing the clonality and entropy of CD8+TILs intransplanted B16 tumors (n=5 for scramble, n=3 for LSD1 KO) asdetermined by TCRseq.

FIG. 10E is volcano and M-A plots showing differentially expressed genesin GFP-labeled B16 tumor cells (n=3 for scramble and LSD1 KO) isolatedfrom tumor-beating immunocompetent mice (referred to as ex vivo cellshereafter), as determined by RNA-seq. Dots in grey representsignificantly increased or decreased genes (FDR<0.05) in LSD1 KO versusscramble cells.

FIG. 10F is volcano and M-A plots showing differentially expressed genesin GFP-labeled B16 tumor cells (n=3 for scramble and LSD1/MDA5 DKO)isolated from tumor-beating immunocompetent mice (referred to as ex vivocells hereafter), as determined by RNA-seq. Dots in grey representsignificantly increased or decreased genes (FDR<0.05) in LSD1/MDA5 DKOversus scramble cells.

FIG. 10G is a heatmap showing differential expression (FDR<0.05) of ERVsbetween scramble and LSD1 KO cells (n=3).

FIG. 10H is plots showing LSD1 and H3K4me2 ChIP-seq signals at genomicloci of 74 ERV subfamilies in control and LSD1 KD cells in ex vivoscramble and LSD1 KO B16 cells.

FIG. 10I is plots showing LSD1 and H3K4me2 ChIP-seq signals at genomicloci of a representative ERVK10C subfamily in control and LSD1 KD cellsin ex vivo scramble and LSD1 KO B16 cells.

FIG. 10J is a representative dotmap showing the top 10 terms of a GOanalysis of upregulated genes (log 2(FC)>1 and FDR <0.05) in LSD1 KOversus control scramble B16 cells. Dot size represents odds ratio.

FIG. 10K is GSEA analysis for inflammatory response in ex vivo LSD1 KOversus scramble B16 cells.

FIG. 10L is a representative box and whisker plot showing log 2(FC) ofupregulated genes in the top 10 terms (170 in total) in LSD1 KO andLSD1.MDA5 DKO versus scramble B16 cells.

FIG. 10M is GSEA analysis for positive regulation of cell proliferationin ex vivo LSD1 KO versus scramble B16 cells.

FIG. 10N is a heatmap showing all genes categorized in GO term “MHCprotein complex”.

FIG. 10O is a bar graph showing mean fluorescent intensity (MFI) ofMHC-1⁺ B16 cells isolated from scramble, LSD1 KO and LSD1/MDA5 DKO B16tumors from immunocompetent mice. Data represents two independentexperiments. Error bars represent SEM of individual mice in oneexperiment. *p<0.05, ***p<0.001, ****p<0.0001, ns, not significant, asdetermined by unpaired t-test.

FIG. 10P is a line graph showing tumor growth of immunocompetent miceinoculated with 250k scramble D4m cells or LSD1 KO D4m cells. Error barsrepresent SEM of individual mice in one experiment. Data represents twoindependent experiments. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001,ns, not significant, as determined by ANOVA.

FIG. 10Q is a line graph showing survival of immunocompetent miceinoculated with 250k scramble D4m cells or LSD1 KO D4m cells. Error barsrepresent SEM of individual mice in one experiment. ****p<0.001,****p<0.0001, ns, not significant, as determined by log-rank test.

FIG. 10R is bar graphs showing the number of CD4⁺ and CD8⁺ TILs per gramof D4m tumor in immunocompetent mice (n=3 in each group) as determinedby flow cytometry. Data represents two independent experiments. Errorbars represent SEM of individual mice in one experiment. *p<0.05,***p<0.001, ****p<0.0001, ns, not significant, as determined by unpairedt-test.

FIG. 10S is a bar graph showing mean fluorescent intensity (MFI) ofMHC-1+ex vivo D4m cells (n=3). Data represents two independentexperiments. Error bars represent SEM of individual mice in oneexperiment. *p<0.05, ***p<0.001, ****p<0.0001, ns, not significant, asdetermined by unpaired t-test.

FIG. 10T is a bar graph showing counts per million (CPM) of PD-L1 oftumor-extracted B16 cells (ex vivo; n=3) as determined by RNA-seq. Datarepresents two independent experiments. Error bars represent SEM ofindividual mice in one experiment. *p<0.05, ***p<0.001, ****p<0.0001,ns, not significant, as determined by unpaired t-test.

FIG. 10U is a bar graph showing MFI of PD-L1⁺ B16 cells isolated fromscramble, LSD1 KO and LSD1/MDA5 DKO B16 tumors from immunocompetentmice. Data represents two independent experiments. Error bars representSEM of individual mice in one experiment. *p<0.05, ***p<0.001,****p<0.0001, ns, not significant, as determined by unpaired t-test.

FIG. 11A is a line graph showing tumor growth of immunocompetent miceinoculated with 250k scramble B16 cells or LSD1 KO B16 cells, andtreated with PD-1 blocking antibody or isotype control based on a settime (day 14) for initial treatment. Arrow bars indicate time points ofanti-PD-1 injection. Error bars represent SEM of individual mice in oneexperiment. Data represents two independent experiments. *p<0.05,**p<0.01, ***p<0.001, ****p<0.0001, ns, not significant, as determinedby ANOVA.

FIG. 11B is a line graph showing survival of immunocompetent miceinoculated with 250k scramble B16 cells or LSD1 KO B16 cells, andtreated with PD-1 blocking antibody or isotype control based on a settime (day 14) for initial treatment. Error bars represent SEM ofindividual mice in one experiment. ****p<0.001, ****p<0.0001, ns, notsignificant, as determined by log-rank test.

FIG. 11C is a line graph showing tumor growth of immunocompetent miceinoculated with 500k scramble B16 cells or LSD1 KO B16 cells, andtreated with PD-1 blocking antibody or isotype control based on a settumor size (˜200 mm³) for initial treatment. Arrow bars indicate timepoints of initial anti-PD-1 injection (black arrow (at day 14)—intoscramble tumor-bearing mice; grey arrow (at day 18)—into LSD1 KOtumor-bearing mice), followed by continuous injection every other dayuntil the end of experiment. Error bars represent SEM of individual micein one experiment. Data represents two independent experiments. *p<0.05,**p<0.01, ***p<0.001, ****p<0.0001, ns, not significant, as determinedby ANOVA.

FIG. 11D is a line graph showing survival of immunocompetent miceinoculated with 500k scramble B16 cells or LSD1 KO B16 cells, andtreated with PD-1 blocking antibody or isotype control based on a settumor size (˜200 mm³) for initial treatment. Error bars represent SEM ofindividual mice in one experiment. ****p<0.001, ****p<0.0001, ns, notsignificant, as determined by log-rank test.

FIG. 12A is bar graph showing the frequencies of mutation, amplificationand deletion of LSD1 across a panel of cancer types were analyzed usingcBioPortal by selecting all listed studies.

FIG. 12B is a representative plot showing analysis of LSD1 RNAexpression in cancerous tissues versus normal tissues from various typesof cancer patients in The Cancer Genome Atlas (TCGA) dataset.

FIG. 12C is survival curves of LSD1-low and LSD1-high patient groupsdichotomously divided by LSD1 median in two cancer types using TCGAdataset.

FIG. 12D is a representative correlation analysis for LSD1 expressionversus IFN/antiviral response in cancerous tissues from various types ofcancer patients in The Cancer Genome Atlas (TCGA) dataset.

FIG. 12E is a representative correlation analysis for LSD1 expressionversus CD8⁺ T cell infiltration in cancerous tissues from various typesof cancer patients in The Cancer Genome Atlas (TCGA) dataset.

FIG. 12F is survival curves of LSD1-low (first tertile, n=113) andLSD1-int/high (second and third tertiles, n=210) SKCM patient groupsdivided based on LSD1 expression using TCGA dataset.

FIG. 12G is a plot of top 10 GO terms based on p value generated byDAVID functional annotation of differentially expressed genes (FC >1.5or FC<0.67, and FDR <0.05) in LSD1-low group versus LSD1-int/high groupof SKCM (increased genes—black (top), decreased genes—grey (bottom)).

FIG. 12H is a plot comparing CD8a expression between LSD1-low group andLSD1-intermediate (int)/high group of SKCM.

FIG. 12I is a plot comparing GzmB expression between LSD1-low group andLSD1-intermediate (int)/high group of SKCM.

DETAILED DESCRIPTION

Chromatin regulators play a broad role in regulating gene expression.When gene regulation goes awry, this can lead to the development ofcancer. Without wishing to be bound by theory, the present disclosuredemonstrated that ablation of the histone demethylase lysine-specificdemethylase 1A (LSD1) in human and mouse cells leads to double-strandedRNA (dsRNA) stress, through elevating the transcript level of certainrepetitive elements and impairing the small RNA machinery, i.e.,RNA-induced silencing complex (RISC), which triggers type I interferonactivation. Significantly, LSD1 deletion in mouse B16 melanoma cellsleads to the activation of potent anti-tumor adaptive immunity, whichrestrained tumor growth in vivo. Importantly, LSD1 depletion alsoelicited dramatic responses of checkpoint blockade-refractory B16 tumorsto anti-PD-1 therapy. The present disclosure describes the potent impactof LSD1 on tumor responses to host immunity and immunotherapy anddescribes LSD1 inhibition combined with PD-1 and/or PD-L1 (sometimescollectively referred to herein as “PD-(L)1”) blockade as a strategy forcancer treatment.

Cancer immunotherapy, including anti-PD(L)1 therapy, has achieved tosuccessful clinical outcomes in controlling tumor progression (Sharmaand Allison (2015) Cell 161(2): 205-214). Recent human clinical trialusing PD-1 or PD-L1 directed immunotherapy have reported promisingresults, leading to FDA approval of PD-1 pathway inhibitors for multipletumor types including melanoma, non-small cell lung cancer (NSCLC), headand neck squamous cell carcinoma (HNSCC), renal cell carcinoma (RCC),Hodgkin's lymphoma, bladder cancer, Merkel cell carcinoma, andmicroinstability high (MS1^(hi)) or mismatch repair deficient adult andpediatric solid tumors (Pauken and Sharpe (2018) Nat Rev Immunol 18(3):153-167). However, a majority of cancer patients do not respond toanti-PD-(L)-1 therapy, due to multiple mechanisms includingdysfunctional T cells and lack of T cell infiltration or recognition byT cells (Sharma et al. (2017) Cell 168: 707-723). The broad roles ofchromatin regulators in controlling cancer and T cell functions raisethe possibility of their involvement in regulating tumor responses orresistance to immunotherapy. Indeed, a recent study found thatinhibition of DNA methylation leads to tumor interferon pathwayactivation, and increased responses to cancer immunotherapy(Chiappinelli et al. (2015) Cell 162(5): 974-986). On the other hand,blocking de novo DNA methylation in T cells enhances anti-PD-L1-mediatedT cells rejuvenation and tumor control (Ghoneim et al. (2017) Cell170:142-157 e119). However, how the full spectrum of chromatinregulators regulates cancer cells and impacts their responses to theimmune system are still poorly understood. Moreover, the therapeuticpotential of manipulating these factors to remodel the cancer chromatinlandscape for onco-immunotherapy is under-explored.

Naturally occurring dsRNAs derived from a variety of sources includingretrotransposons are processed into endo-siRNA by RISC (Watanabe et al.(2008) Nature 453(7194): 539-543). The epigenetic regulation ofretrotransposon (such as ERVs) transcription in mammal germ cells andearly embryonic development are well documented (Leung and Lorincz(2012) Trends Biochem Sci 37(4): 127-133; Song et al. (2012) Nat Rev MolCell Biol 13(5): 283-296), but much less is known in differentiatedsomatic cells. Without wishing to be bound by theory, the presentdisclosure shows that LSD1 represses the transcription of a subset ofERVs in human cancer cells, consistent with a previous report showingLSD1 is involved in regulating ERV expression in mESCs (Macfarlan et al.(2011) Genes Dev 25(6): 594-607). Although ERV transcript induction byLSD1 inhibition is not dramatic, their dsRNA forms are much moresignificantly elevated. Additionally, intracellular dsRNAs can bederived from different categories of transcripts, including ERVs, LINES,SINes and gene/pseudogene duplexes (Carthew and Sontheimer 2009 Cell136: 642-655), many of which are also up-regulated in the LSD1 nulltumor cells. Importantly, it's the dsRNA forms, rather than the overalltranscripts, that are directly recognized by dsRNA sensors to induce IFNactivation. LSD1 inhibition also compromises the expression of RISCproteins and subsequently RISC activity, thus blocking dsRNA fromentering the RNA interference pathway. By coordinating these twoprocesses, LSD1 inhibition reinforces dsRNA stress and subsequentcellular responses.

Non-limiting aspects of these methods are described below, and can beused in any combination without limitation. Additional aspects of thesemethods are known in the art.

Methods of Treatment

Provided herein are methods of treating cancer in a patient. Exemplarymethods include administering to a patient in need of cancer treatmenttherapeutically effective amounts of a lysine-specific demethylase 1A(LSD1) inhibitor and a programmed-cell death 1 (PD-1) inhibitor or aprogrammed-cell death ligand 1 (PD-L1) inhibitor, or both, to therebytreat cancer in the patient.

Also provided herein are methods of treating cancer in a patient thatinclude, e.g. administering to a patient in need of cancer treatmenttherapeutically effective amounts of a lysine-specific demethylase 1A(LSD1) inhibitor and at least one immunotherapy other than a PD-1 orPD-L1 inhibitor, to thereby treat cancer in the patient.

In methods described herein, the cancer can be, e.g., a primary tumor, ametastatic tumor, or a non-T-cell-infiltrating tumor.

In any of the presently-described methods, the cancer can be, e.g.,melanoma, acute myeloid leukemia (AML), squamous cell carcinoma, renalcell carcinoma, non-small cell lung cancer (NSCLC), small cell lungcancer (SCLC), gastric cancer, bladder cancer, kidney cancer, head andneck cancer, Ewing sarcoma, Hodgkin's lymphoma, Merkel cell carcinoma,breast cancer or prostate cancer. Treatment of multiple cancer types atthe same time is contemplated by and within the present disclosure.

A cancer described herein can be, e.g., a PD-1 and/or PD-L1 refractoryor resistant cancer. In some instances, the patient having the cancermay have previously received cancer treatment (e.g., any of the cancertreatment described herein).

Administering may be performed, e.g., at least once (e.g., at least2-times, at least 3-times, at least 4-times, at least 5-times, at least6-times, at least 7-times, at least 8-times, at least 9-times, at least10-times, at least 11-times, at least 12-times, at least 13-times, or atleast 14-times) a week. Also contemplated are monthly treatments, e.g.administering at least once per month for at least 1 month (e.g., atleast two, three, four, five, or six or more months, e.g., 12 or moremonths), and yearly treatments (e.g., administration once a year for oneor more years). Administration can be via any art-known means, e.g.,intravenous, subcutaneous, intraperitoneal, oral, and/or rectaladministration, or any combination of known administration methods.

Administration can include administering compositions in any usefulformat. For example, skilled practitioners will appreciate that a numberof compositions are within the present invention. One useful compositionmay be a combination composition comprising an LSD1 inhibitor and a PD-1and/or PD-L1 inhibitor. Such a combined composition can be administeredto the patient in any useful dosing regimen. When using separatecompositions, e.g., a first composition comprising an LSD1 inhibitor anda second composition comprising a PD-1 and/or PD-L1 inhibitor, thecompositions can be administered in any order. For example, the firstcomposition can be administered followed by administration of the secondcomposition, or the second composition can be administered before thefirst composition, or the first and second compositions can beadministered essentially simultaneously.

In one aspect of any of the methods described herein, a firstcomposition comprising an LSD1 inhibitor is administered prior to theadministration of a second composition comprising a PD-1 and/or PD-L1inhibitor. For example, a patient can receive at least one dose (e.g.,at least two doses, at least three doses, at least four doses, at leastfive doses, at least six doses, at least seven doses, at least eightdoses, at least nine doses, at least ten doses, at least eleven doses,or at least twelve doses) of first composition comprising an LSD1inhibitor prior to the administration of a second composition comprisinga PD-1 and/or PD-L1 inhibitor.

As used herein, treating includes “prophylactic treatment”, which meansreducing the incidence of or preventing (or reducing the risk of) a signor symptom of a cancer in a patient at risk of developing a cancer. Theterm “therapeutic treatment” refers to reducing signs or symptoms of acancer, reducing cancer progression, reducing severity of a cancer,and/or re-occurrence in a cancer patient.

The methods described herein may in some instances include administeringa composition, e.g., a sterile composition, comprising an inhibitorynucleic acid that is complementary to LSD1 or PD-1 as described herein.A composition may include a LSD1 inhibitory nucleic acid or a PD-1inhibitory nucleic acid, or both. Inhibitory nucleic acids for use inpracticing the methods described herein are described below.

Inhibitory nucleic acids have been employed as therapeutic moieties inthe treatment of disease states in subjects, including humans.Inhibitory nucleic acids can be useful therapeutic modalities that canbe configured to be useful in treatment regimens for the treatment ofcells, tissues and animals, especially humans.

For therapeutics, a subject, e.g., a human, having cancer or suspectedof having cancer, or at increased risk of developing a cancer (e.g., byvirtue of family history, genetic testing, or presence of otheridentified risk factor), can be treated by administering an inhibitorynucleic acid in accordance with this disclosure. For example, in onenon-limiting embodiment, the methods comprise the step of administeringto the subject in need of treatment a therapeutically effective amountof one or more of a LSD1 inhibitory nucleic acid (e.g., a LSD1 antisensemolecule, a LSD1 small interfering RNA, a LSD1 small hairpin RNA), aPD-1 inhibitory nucleic acid (e.g., a PD-1 antisense molecule, a PD-1small interfering RNA, a PD-1 small hairpin RNA) or a PD-L1 inhibitorynucleic acid (e.g., a PD-L1 antisense molecule, a PD-L1 smallinterfering RNA, a PD-L1 small hairpin RNA) as described herein.

Immunotherapy

An immunotherapy can be administered to the patient in methods describedherein. The term “immunotherapy” refers to a therapeutic treatment thatinvolves administering to a patient an agent that modulates the immunesystem. For example, an immunotherapy can increase the expression and/oractivity of a regulator of the immune system. In other instances, animmunotherapy can decrease the expression and/or activity of a regulatorof the immune system. In some instances, an immunotherapy can recruitand/or enhance the activity of an immune cell. An example of animmunotherapy is a therapeutic treatment that involves administering atleast one, e.g., two or more, immune checkpoint inhibitors. Exemplaryimmune checkpoint inhibitors useful in the presently-described methodsare CTLA-4 inhibitors, PD-1 inhibitors, PD-L1 inhibitors, PD-L2inhibitors, OX40 inhibitor, TIM3 inhibitors, or LAG3 inhibitors, orcombinations thereof.

The immunotherapy can be a cellular immunotherapy (e.g., adoptive T-celltherapy, dendritic cell therapy, natural killer cell therapy). Forexample, the cellular immunotherapy can be sipuleucel-T (APC8015;Provenge™; Plosker (2011) Drugs 71(1): 101-108). In some instances, thecellular immunotherapy includes cells that express a chimeric antigenreceptor (CAR). In some instances, the cellular immunotherapy can be aCAR-T cell therapy, e.g., tisagenlecleucel (Kymriah™).

Immunotherapy can be, e.g., an antibody therapy (e.g., a monoclonalantibody, a conjugated antibody). In some embodiments, the antibody isan anti-CTLA-4 antibody, an anti-PD-1 antibody, an anti-PD-L1 antibody,an anti-PD-L2 antibody, an anti-OX40 antibody, an anti-TIM3 antibody, oran anti-LAG3 antibody. Exemplary antibody therapies are bevacizumab(Mvasti™, Avastin®), trastuzumab (Herceptin®), avelumab (Bavencio®),rituximab (MabThera™, Rituxan®), edrecolomab (Panorex), daratumuab(Darzalex®), olaratumab (Lartruvo™) ofatumumab (Arzerra®), alemtuzumab(Campath®), cetuximab (Erbitux®), oregovomab, pembrolizumab (Keytruda®),dinutiximab (Unituxin®), obinutuzumab (Gazyva®), tremelimumab(CP-675,206), ramucirumab (Cyramza®), ublituximab (TG-1101), panitumumab(Vectibix®), elotuzumab (Empliciti™), avelumab (Bavencio®), necitumumab(Portrazza™), cirmtuzumab (UC-961), ibritumomab (Zevalin®), isatuximab(SAR650984), nimotuzumab, fresolimumab (GC1008), lirilumab (INN),mogamulizumab (Poteligeo®), ficlatuzumab (AV-299), denosumab (Xgeva®),ganitumab, urelumab, pidilizumab or amatuximab.

An immunotherapy described herein can involve administering anantibody-drug conjugate to a patient. The antibody-drug conjugate canbe, e.g., gemtuzumab ozogamicin (Mylotarg™), inotuzumab ozogamicin(Besponsa®), brentuximab vedotin (Adcetris®), ado-trastuzumab emtansine(TDM-1; Kadcyla®), mirvetuximab soravtansine (IMGN853) or anetumabravtansine.

In some instances, the immunotherapy includes blinatumomab (AMG103;Blincyto®) or midostaurin (Rydapt).

An immunotherapy can include administering to the patient a toxin. Forexample, the immunotherapy can including administering denileukindiftitox (Ontak®).

In some instances, the immunotherapy can be a cytokine therapy. Thecytokine therapy can be, e.g., an interleukin 2 (IL-2) therapy, aninterferon alpha (IFN-α) therapy, a granulocyte colony stimulatingfactor (G-CSF) therapy, an interleukin 12 (IL-12) therapy, aninterleukin 15 (IL-15) therapy, an interleukin 7 (IL-7) therapy or anerythropoietin-alpha (EPO) therapy. In some embodiments, the IL-2therapy is aldesleukin (Proleukin®). In some embodiments, the IFN-αtherapy is IntronA® (Roferon-A®). In some embodiments, the G-CSF therapyis filgrastim (Neupogen®).

In some instances, the immunotherapy is an immune checkpoint inhibitor.For example, the immunotherapy can include administering one or moreimmune checkpoint inhibitors. In some embodiments, the immune checkpointinhibitor is a CTLA-4 inhibitor, a PD-1 inhibitor or a PD-L1 inhibitor.An exemplary CTLA-4 inhibitor would be, e.g., ipilimumab (Yervoy®) ortremelimumab (CP-675,206). In some embodiments, the PD-1 inhibitor ispembrolizumab (Keytruda®) or nivolumab (Opdivo®). In some embodiments,the PD-L1 inhibitor is atezolizumab (Tecentriq®), avelumab (Bavencio®)or durvalumab (Imfinzi™).

In some instances, the immunotherapy is mRNA-based immunotherapy. Forexample, the mRNA-based immunotherapy can be CV9104 (see, e.g., Rauschet al. (2014) Human Vaccin Immunother 10(11): 3146-52; and Kubler et al.(2015) J. Immunother Cancer 3:26).

In some instances, the immunotherapy can involve bacillusCalmette-Guerin (BCG) therapy.

In some instances, the immunotherapy can be an oncolytic virus therapy.For example, the oncolytic virus therapy can involve administeringtalimogene alherparepvec (T-VEC; Imlygic®).

In some instances, the immunotherapy is a cancer vaccine, e.g., a humanpapillomavirus (HPV) vaccine. For example, an HPV vaccine can beGardasil®, Gardasil9® or Cervarix®. In some instances, the cancervaccine is a hepatitis B virus (HBV) vaccine. In some embodiments, theHBV vaccine is Engerix-B®, Recombivax HB® or GI-13020 (Tarmogen®). Insome embodiments, the cancer vaccine is Twinrix® or Pediarix®. In someembodiments, the cancer vaccine is BiovaxlD®, Oncophage®, GVAX,ADXS11-001, ALVAC-CEA, PROSTVAC®, Rindopepimut®, CimaVax-EGF,lapuleucel-T (APC8024; Neuvenge™), GRNVAC1, GRNVAC2, GRN-1201,hepcortespenlisimut-L (Hepko-V5), DCVAX®, SCIB1, BMT CTN 1401, PrCaVBIR, PANVAC, ProstAtak®, DPX-Survivac, or viagenpumatucel-L (HS-110).

The immunotherapy can involve, e.g., administering a peptide vaccine.For example, the peptide vaccine can be nelipepimut-S(E75) (NeuVax™),IMA901, or SurVaxM (SVN53-67). In some instances, the cancer vaccine isan immunogenic personal neoantigen vaccine (see, e.g., Ott et al. (2017)Nature 547: 217-221; Sahin et al. (2017) Nature 547: 222-226). In someembodiments, the cancer vaccine is RGSH4K, or NEO-PV-01. In someembodiments, the cancer vaccine is a DNA-based vaccine. In someembodiments, the DNA-based vaccine is a mammaglobin-A DNA vaccine (see,e.g., Kim et al. (2016) OncoImmunology 5(2): e1069940).

Cancer

The methods described herein can be used in cancer treatments.Non-limiting examples of cancer include: acute lymphoblastic leukemia(ALL), acute myeloid leukemia (AML), adrenocortical carcinoma, analcancer, appendix cancer, astrocytoma, basal cell carcinoma, brain tumor,bile duct cancer, bladder cancer, bone cancer, breast cancer, bronchialtumor, Burkitt Lymphoma, carcinoma of unknown primary origin, cardiactumor, cervical cancer, chordoma, chronic lymphocytic leukemia (CLL),chronic myelogenous leukemia (CML), chronic myeloproliferative neoplasm,colon cancer, colorectal cancer, craniopharyngioma, cutaneous T-celllymphoma, ductal carcinoma, embryonal tumor, endometrial cancer,ependymoma, esophageal cancer, esthesioneuroblastoma, fibroushistiocytoma, Ewing sarcoma, eye cancer, germ cell tumor, gallbladdercancer, gastric cancer, gastrointestinal carcinoid tumor,gastrointestinal stromal tumor, gestational trophoblastic disease,glioma, head and neck cancer, hairy cell leukemia, hepatocellularcancer, histiocytosis, Hodgkin lymphoma, hypopharyngeal cancer,intraocular melanoma, islet cell tumor, Kaposi sarcoma, kidney cancer,Langerhans cell histiocytosis, laryngeal cancer, leukemia, lip and oralcavity cancer, liver cancer, lobular carcinoma in situ, lung cancer,lymphoma, macroglobulinemia, malignant fibrous histiocytoma, melanoma,Merkel cell carcinoma, mesothelioma, metastatic squamous neck cancerwith occult primary, midline tract carcinoma involving NUT gene, mouthcancer, multiple endocrine neoplasia syndrome, multiple myeloma, mycosisfungoides, myelodysplastic syndrome, myelodysplastic/myeloproliferativeneoplasm, nasal cavity and para-nasal sinus cancer, nasopharyngealcancer, neuroblastoma, non-Hodgkin lymphoma, non-small cell lung cancer,oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer,papillomatosis, paraganglioma, parathyroid cancer, penile cancer,pharyngeal cancer, pheochromocytomas, pituitary tumor, pleuropulmonaryblastoma, primary central nervous system lymphoma, prostate cancer,rectal cancer, renal cell cancer, renal pelvis and ureter cancer,retinoblastoma, rhabdoid tumor, salivary gland cancer, Sezary syndrome,skin cancer, small cell lung cancer, small intestine cancer, soft tissuesarcoma, spinal cord tumor, stomach cancer, T-cell lymphoma, teratoidtumor, testicular cancer, throat cancer, thymoma and thymic carcinoma,thyroid cancer, urethral cancer, uterine cancer, vaginal cancer, vulvarcancer, and Wilms' tumor.

For example, any of the methods described herein can be used to treat acancer selected from the group consisting of: melanoma, acute myeloidleukemia (AML), squamous cell carcinoma, renal cell carcinoma, non-smallcell lung cancer (NSCLC), small cell lung cancer (SCLC), gastric cancer,bladder cancer, kidney cancer, head and neck cancer, Ewing sarcoma,Hodgkin's lymphoma, Merkel cell carcinoma, breast cancer and prostatecancer.

LSD1

As used herein, the term “LSD1 inhibitor” refers to a therapeutic agentthat reduces, decreases, blocks or inhibits the expression or activityof LSD1. For example, the LSD1 inhibitor can block or disrupt thecatalytic active site of LSD1. The LSD1 inhibitor can be, e.g., aselective LSD1 inhibitor or a non-selective LSD1 inhibitor.

The LSD1 inhibitor can be a small molecule, an antibody, or aninhibitory nucleic acid. A non-exhaustive list of small molecule LSD1inhibitors is provided in Table 1.

TABLE 1 Exemplary list of small molecule LSD1 inhibitors Generic name orname as used in Chemical name clinical trials CAS#trans-2-Phenylcyclopropylamine Tranylcypromine 13492-01-8 hemisulfatesalt 1-(4-methylpiperazin-1-yl)-2-[[2-(4- RN 1 1781835-13-9phenylmethoxyphenyl)cyclopropyl] dihydrochloride amino]ethanonerel-N-[(1R,2S)-2-Phenylcyclopropyl]-4- GSK-LSD1 1431368-48-7Piperidinamine hydrochloride (1:2) 4-[4-[[[(1R,2S)-2- GSK28795521401966-69-5 phenylcyclopropyl]amino]methyl]-1-piperidinyl]methyl]-benzoic acid rel-N1-[(1R,2S)-2-phenylcyclopropyl]-1,ORY1001 1431326-61-2 4-cyclohexanediamine, dihydrochloride(R)-4-[5-(Pyrrolidin-3-ylmethoxy)-2-p- GSK690 2101305-84-2tolyl-pyridin-3-yl]-benzonitrile Bis-TFA Salt3-Chloro-6-nitro-2-(trifluoromethyl)-4H- Namoline 342795-11-31-benzopyran-4-one 1,15-bis{N5-[3,3-(diphenyl)propyl]-N1- Cpd 2dbiguanido}-4,12-diazapentadecane (1R,2S)-rel-2-[3,5-Difluoro-2- S21011239262-36-2 (phenylmethoxy)phenyl]cycloprpanamine hydrochloride4′-[(1R,2S)-2-aminocyclopropyl]-[1,1′- OG-L002 1357302-64-7bipheny1]-3-ol 3-(4-morpholinylsulfonyl)-benzoic acid SP25091423715-09-6 (2E)-2-[1-(5-chloro-2- hydroxyphenyl)ethylidene]hydrazideMethyl-3-(4-(4- CBB1007 1379573-92-8 carbamimidoylbenzoyl)piperazine-1-carbonyl)-5-((4-carbamimidoylpiperazin- 1-yl)methyl)benzoateN-[(2S)-5-{[(1R, 2S)-2-(4- IMG-7289fluorophenyl)cyclopropyl]amino}-1-(4-methylpiperazin-1-yl)-1-oxopentan-2-y1]-4-(1H-1,2,3-triazol-1-yl)benzamide, bistosylate salt

In some instances, the LSD1 inhibitor is an inhibitory nucleic acid. SEQID NO: 1 is an exemplary human sequence of LSD1:

(SEQ ID NO: 1; Accession Number: NM_001009999.2)a tgttatctgg gaagaaggcg gcagccgcgg 181cggcggcggc tgcagcggca gcaaccggga cggaggctgg ccctgggaca gcaggcggct 241ccgagaacgg gtctgaggtg gccgcgcagc ccgcgggcct gtcgggccca gccgaggtcg 301ggccgggggc ggtgggggag cgcacacccc gcaagaaaga gcctccgcgg gcctcgcccc 361ccgggggcct ggcggaaccg ccggggtccg cagggcctca ggccggccct actgtcgtgc 421ctgggtctgc gacccccatg gaaactggaa tagcagagac tccggagggg cgtcggacca 481gccggcgcaa gcgggcgaag gtagagtaca gagagatgga tgaaagcttg gccaacctct 541cagaagatga gtattattca gaagaagaga gaaatgccaa agcagagaag gaaaagaagc 601ttcccccacc accccctcaa gccccacctg aggaagaaaa tgaaagtgag cctgaagaac 661catcggggca agcaggagga cttcaagacg acagttctgg agggtatgga gacggccaag 721catcaggtgt ggagggcgca gctttccaga gccgacttcc tcatgaccgg atgacttctc 781aagaagcagc ctgttttcca gatattatca gtggaccaca acagacccag aaggtttttc 841ttttcattag aaaccgcaca ctgcagttgt ggttggataa tccaaagatt cagctgacat 901ttgaggctac tctccaacaa ttagaagcac cttataacag tgatactgtg cttgtccacc 961gagttcacag ttatttagag cgtcatggtc ttatcaactt cggcatctat aagaggataa 1021aacccctacc aactaaaaag acaggaaagg taattattat aggctctggg gtctcaggct 1081tggcagcagc tcgacagtta caaagttttg gaatggatgt cacacttttg gaagccaggg 1141atcgtgtggg tggacgagtt gccacatttc gcaaaggaaa ctatgtagct gatcttggag 1201 ccatggtggt aacaggtctt ggagggaatc ctatggctgt ggtcagcaaa caagtaaata 1261tggaactggc caagatcaag caaaaatgcc cactttatga agccaacgga caagctgaca 1321ctgtcaaggt tcctaaagag aaagatgaaa tggtagagca agagtttaac cggttgctag 1381aagctacatc ttaccttagt catcaactag acttcaatgt cctcaataat aagcctgtgt 1441cccttggcca ggcattggaa gttgtcattc agttacaaga gaagcatgtc aaagatgagc 1501agattgaaca ttggaagaag atagtgaaaa ctcaggaaga attgaaagaa cttcttaata 1561agatggtaaa tttgaaagag aaaattaaag aactccatca gcaatacaaa gaagcatctg 1621aagtaaagcc acccagagat attactgccg agttcttagt gaaaagcaaa cacagggatc 1681tgaccgccct atgcaaggaa tatgatgaat tagctgaaac acaaggaaag ctagaagaaa 1741aacttcagga gttggaagcg aatcccccaa gtgatgtata tctctcatca agagacagac 1801aaatacttga ttggcatttt gcaaatcttg aatttgctaa tgccacacct ctctcaactc 1861tctcccttaa gcactgggat caggatgatg actttgagtt cactggcagc cacctgacag 1921taaggaatgg ctactcgtgt gtgcctgtgg ctttagcaga aggcctagac attaaactga 1981atacagcagt gcgacaggtt cgctacacgg cttcaggatg tgaagtgata gctgtgaata 2041cccgctccac gagtcaaacc tttatttata aatgcgacgc agttctctgt acccttcccc 2101tgggtgtgct gaagcagcag ccaccagccg ttcagtttgt gccacctctc cctgagtgga 2161aaacatctgc agtccaaagg atgggatttg gcaaccttaa caaggtggtg ttgtgttttg 2221atcgggtgtt ctgggatcca agtgtcaatt tgttcgggca tgttggcagt acgactgcca 2281gcaggggtga gctcttcctc ttctggaacc tctataaagc tccaatactg ttggcactag 2341tggcaggaga agctgctggt atcatggaaa acataagtga cgatgtgatt gttggccgat 2401gcctggccat tctcaaaggg atttttggta gcagtgcagt acctcagccc aaagaaactg 2461tggtgtctcg ttggcgtgct gatccctggg ctcggggctc ttattcctat gttgctgcag 2521gatcatctgg aaatgactat gatttaatgg ctcagccaat cactcctggc ccctcgattc 2581caggtgcccc acagccgatt ccacgactct tctttgcggg agaacatacg atccgtaact 2641acccagccac agtgcatggt gctctgctga gtgggctgcg agaagcggga agaattgcag 2701accagttttt gggggccatg tatacgctgc ctcgccaggc cacaccaggt gttcctgcac 2761agcagtcccc aagcatgtga

Inhibitory nucleic acids useful in the present methods and compositionsinclude those that are designed to inhibit LSD1. SEQ ID NO: 2 is anexemplary shRNA sequence that targets human LSD1:5′-CCGG-GCCTAGACATTAAACTGAATA-CTCGAG-TATTCAGTTTAATGTCTAGGC-TTTTTG-3′(SEQ ID NO: 2). Bold and underlined portions are targeting/matchingsequences in human LSD1 mRNA.

The LSD1 inhibitory nucleic acid can, e.g., comprise SEQ ID NO: 2. Forexample, the LSD1 inhibitory nucleic acid can be a nucleic acidcomprising a sequence that is complementary to a contiguous sequence ofat least 5 (e.g., at least 6, at least 7, at least 8, at least 9, atleast 10, at least 11, at least 12, at least, 13, at least 14, at least15, at least 16, at least 17, at least 18, at least 19, at least 20, atleast 21, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,or 22) nucleotides present in SEQ ID NO: 2.

The LSD1 inhibitory nucleic acid can be any LSD1 inhibitory nucleic acidthat decreases, reduces or silences the expression and/or activity ofLSD1. As shown herein, loss of LSD1 increased the expression of HERV-E,HERV-K, HML-2, ERVL, IFN-α, IFN-β, IL-28, ISG15, OASL, RIG-I, TLR3, andMDA-5. In some embodiments, the LSD1 inhibitory nucleic acid is any LSD1inhibitory nucleic acid that increases or upregulates the expressionand/or activity of HERV-E, HERV-K, HML-2, ERVL, IFN-α, IFN-β, IL-28,ISG15, OASL, RIG-I, TLR3, and/or MDA-5.

In some embodiments, the LSD1 inhibitor can be a compound having thestructure of Formula I, or Formula II, or a pharmaceutically acceptablesalt thereof:

Wherein R₁ is selected from the group consisting of C₁-C₆ alkyl,—NHSO₂Me, —NHSO₂Ph, arylalkoxy, C₃-C₇ cycloalkyl, —NHC(O)R_(a),1-methyl-1H-pyrazol-4-yl, hydroxyl, C₁-C₄alkoxy, halogen, amino,substituted amino, and —C(O)OR_(a);

-   -   R₃ is selected from the group consisting of aryl, heteroaryl        —SO₂R_(a), —NHC(O)R_(a), —CH₂C(O)OR_(a), —C(O)OR_(a),        —C(O)R_(a), —C(O)NR_(a)R_(b), amino, substituted amino,        arylalkyl, and heteroarylalkyl;    -   each R_(a) is independently hydrogen, phenyl, phenylmethyl,        3,5-dimethylisoxazol-4-yl, 1,2-dimethyl-1H-imidazol-4-yl,        C₃-C₇cycloalkyl, or C₁-C₆alkyl;    -   R_(b) is hydrogen or C₁-C₃alkyl; or    -   R_(a) and R_(b) together form a 5- or 6-membered        heterocycloalkyl ring;    -   R₄ is H;    -   W is —(CH₂)₁₋₄ or —CH(R_(c))(CH₂)₀₋₃, in which R_(c) is —CN or        C₁-C₄alkyl;    -   X is N;    -   Z is (CH₂)_(q), wherein q is 0-2, and wherein when q is 0, Z        represents a bond; and m is 0-3; or a pharmaceutically        acceptable salt thereof. A detailed description regarding these        LSD1 inhibitors can be found, e.g., in U.S. Pat. No. 9,346,840,        which is incorporated herein by reference in its entirety.

In some embodiments, the LSD1 inhibitor is an LSD1 inhibitor know in theart, e.g., in US 20150225401, US 20170129857, US20170281567,US20170281566, US20170183308, US20170283397, US20170209432,US20170044101, U.S. Pat. Nos. 9,493,442, 9,346,840, WO/2016/007736,WO/2016/161282, US 20160009711, and Fu et al., Advances toward LSD1inhibitors for cancer therapy, Future Medicinal Chemistry, vol. 9, no.11 (2017)|; each of which is incorporated herein by reference in itsentirety.

PD-1

In some instances, the PD-1 inhibitor is a small molecule, an antibodyor an inhibitory nucleic acid. In some embodiments, the PD-1 antibodycan, e.g., be selected from the group consisting of: nivolumab (Opdivo®)and pembrolizumab (Keytruda®). Numerous anti-PD-1 antibodies are knownin the art, and are described, e.g., in U.S. Pat. No. 9,771,425,US20170240635, US20180030137, U.S. Pat. No. 9,914,783, US20160362489,U.S. Pat. Nos. 9,084,776, 9,102,727, 9,492,540; each of which isincorporated herein by reference in its entirety.

In some instances, the PD-1 inhibitor is an inhibitory nucleic acid. SEQID NO: 3 is an exemplary human sequence of PD-1:

(SEQ ID NO: 3; Accession Number NM_005018.2)at gcagatccca caggcgccct ggccagtcgt ctgggcggtg ctacaactgg 121gctggcggcc aggatggttc ttagactccc cagacaggcc ctggaacccc cccaccttct 181ccccagccct gctcgtggtg accgaagggg acaacgccac cttcacctgc agcttctcca 241acacatcgga gagcttcgtg ctaaactggt accgcatgag ccccagcaac cagacggaca 301agctggccgc cttccccgag gaccgcagcc agcccggcca ggactgccgc ttccgtgtca 361cacaactgcc caacgggcgt gacttccaca tgagcgtggt cagggcccgg cgcaatgaca 421gcggcaccta cctctgtggg gccatctccc tggcccccaa ggcgcagatc aaagagagcc 481tgcgggcaga gctcagggtg acagagagaa gggcagaagt gcccacagcc caccccagcc 541cctcacccag gccagccggc cagttccaaa ccctggtggt tggtgtcgtg ggcggcctgc 601tgggcagcct ggtgctgcta gtctgggtcc tggccgtcat ctgctcccgg gccgcacgag 661ggacaatagg agccaggcgc accggccagc ccctgaagga ggacccctca gccgtgcctg 721tgttctctgt ggactatggg gagctggatt tccagtggcg agagaagacc ccggagcccc 781ccgtgccctg tgtccctgag cagacggagt atgccaccat tgtctttcct agcggaatgg 841gcacctcatc ccccgcccgc aggggctcag ctgacggccc tcggagtgcc cagccactga 901ggcctgagga tggacactgc tcttggcccc tctga

Inhibitory nucleic acids useful in the present methods and compositionsinclude those that are designed to inhibit PD-1. SEQ ID NO: 4 is anexemplary shRNA sequence that targets human PD-1:5′-CCGG-CATTGTCTTTCCTAGCGGAAT-CTCGAG-ATTCCGCTAGGAAAGACAATG-TTTTTG-3′(SEQ ID NO: 4). Bold and underlined portions are targeting/matchingsequences in human PD-1 mRNA.

The PD-1 inhibitory nucleic acid can, e.g., comprise SEQ ID NO: 4. Forexample, the PD-1 inhibitory nucleic acid can be a nucleic acidcomprising a sequence that is complementary to a contiguous sequence ofat least 5 (e.g., at least 6, at least 7, at least 8, at least 9, atleast 10, at least 11, at least 12, at least, 13, at least 14, at least15, at least 16, at least 17, at least 18, at least 19, at least 20, atleast 21, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,or 22) nucleotides present in SEQ ID NO: 4.

PD-L1

The PD-L1 inhibitor can be, e.g., a small molecule, an antibody or aninhibitory nucleic acid.

For example, PD-L1 antibodies useful in the presently described methodsinclude durvalumab (Imfmnzi™), atezolizumab (Tecentriq®) and avelumab(Bavencio®). Numerous anti-PD-L1 antibodies are known in the art, andare described, e.g., in U.S. Pat. No. 9,789,183, US20170319690, U.S.Pat. No. 9,624,298, US20100086550, U.S. Pat. No. 8,617,546,US20180079814; each of which is incorporated herein by reference in itsentirety.

In some instances, the PD-L1 inhibitor is an inhibitory nucleic acid.SEQ ID NO: 5 is an exemplary human sequence of PD-L1:

(SEQ ID NO: 5; Accession Number NM 014143.3) at gaggatattt 121gctgtcttta tattcatgac ctactggcat ttgctgaacg catttactgt cacggttccc 181aaggacctat atgtggtaga gtatggtagc aatatgacaa ttgaatgcaa attcccagta 241gaaaaacaat tagacctggc tgcactaatt gtctattggg aaatggagga taagaacatt 301attcaatttg tgcatggaga ggaagacctg aaggttcagc atagtagcta cagacagagg 361gcccggctgt tgaaggacca gctctccctg ggaaatgctg cacttcagat cacagatgtg 421aaattgcagg atgcaggggt gtaccgctgc atgatcagct atggtggtgc cgactacaag 481cgaattactg tgaaagtcaa tgccccatac aacaaaatca accaaagaat tttggttgtg 541gatccagtca cctctgaaca tgaactgaca tgtcaggctg agggctaccc caaggccgaa 601gtcatctgga caagcagtga ccatcaagtc ctgagtggta agaccaccac caccaattcc 661aagagagagg agaagctttt caatgtgacc agcacactga gaatcaacac aacaactaat 721gagattttct actgcacttt taggagatta gatcctgagg aaaaccatac agctgaattg 781gtcatcccag aactacctct ggcacatcct ccaaatgaaa ggactcactt ggtaattctg 841ggagccatct tattatgcct tggtgtagca ctgacattca tcttccgttt aagaaaaggg 901agaatgatgg atgtgaaaaa atgtggcatc caagatacaa actcaaagaa gcaaagtgat 961acacatttgg aggagacgta a

Inhibitory nucleic acids useful in the present methods and compositionsinclude those that are designed to inhibit PD-L1.

SEQ ID NO: 6 is an exemplary shRNA sequence that targets human PD-L1:5′-CCGG-CTGACATTCATCTTCCGTTTA-CTCGAG-TAAACGGAAGATGAATGTCAG-TTTTTG-3′(SEQ ID NO: 6). Bold and underlined portions are targeting/matchingsequences in human PD-L1 mRNA.

In some embodiments, the PD-L1 inhibitory nucleic acid comprises SEQ IDNO: 6. In some embodiments, the PD-L1 inhibitory nucleic acid is anucleic acid comprising a sequence that is complementary to a contiguoussequence of at least 5 (e.g., at least 6, at least 7, at least 8, atleast 9, at least 10, at least 11, at least 12, at least, 13, at least14, at least 15, at least 16, at least 17, at least 18, at least 19, atleast 20, at least 21, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, or 22) nucleotides present in SEQ ID NO: 6.

The PD-L1 inhibitory nucleic acid can be any PD-L1 inhibitory nucleicacid that decreases, reduces or silences the expression and/or activityof PD-L1. For example, the PD-L-1 inhibitory nucleic acid can be anyPD-L1 inhibitory nucleic acid that decreases, reduces or silences theexpression and/or activity of PD-L1.

Inhibitory Nucleic Acids

Inhibitory nucleic acids useful in the present methods and compositionsinclude antisense oligonucleotides, ribozymes, external guide sequence(EGS) oligonucleotides, siRNA compounds, single- or double-stranded RNAinterference (RNAi) compounds such as siRNA compounds, modifiedbases/locked nucleic acids (LNAs), peptide nucleic acids (PNAs), andother oligomeric compounds or oligonucleotide mimetics which hybridizeto at least a portion of the target nucleic acid and modulate itsfunction. In some embodiments, the inhibitory nucleic acids includeantisense RNA, antisense DNA, chimeric antisense oligonucleotides,antisense oligonucleotides comprising modified linkages, interferenceRNA (RNAi), short interfering RNA (siRNA); a micro, interfering RNA(miRNA); a small, temporal RNA (stRNA); or a short, hairpin RNA (shRNA);small RNA-induced gene activation (RNAa); small activating RNAs(saRNAs), or combinations thereof. See, e.g., WO 2010040112.

In some embodiments, the inhibitory nucleic acids are 10 to 50, 10 to20, 10 to 25, 13 to 50, or 13 to 30 nucleotides in length. One havingordinary skill in the art will appreciate that this embodies inhibitorynucleic acids having complementary portions of 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50nucleotides in length, or any range therewithin. In some embodiments,the inhibitory nucleic acids are 15 nucleotides in length. In someembodiments, the inhibitory nucleic acids are 12 or 13 to 20, 25, or 30nucleotides in length. One having ordinary skill in the art willappreciate that this embodies inhibitory nucleic acids havingcomplementary portions of 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29 or 30 nucleotides in length, or any rangetherewithin (complementary portions refers to those portions of theinhibitory nucleic acids that are complementary to the target sequence).

The inhibitory nucleic acids useful in the present methods aresufficiently complementary to the target RNA, i.e., hybridizesufficiently well and with sufficient specificity, to give the desiredeffect. “Complementary” refers to the capacity for pairing, throughhydrogen bonding, between two sequences comprising naturally ornon-naturally occurring bases or analogs thereof. For example, if a baseat one position of an inhibitory nucleic acid is capable of hydrogenbonding with a base at the corresponding position of a RNA, then thebases are considered to be complementary to each other at that position.100% complementarity is not required.

Routine methods can be used to design an inhibitory nucleic acid thatbinds to the target sequence (e.g., LSD1, PD-1, PD-L1, PD-L2, OX40,TIM3, LAG3) with sufficient specificity. In some embodiments, themethods include using bioinformatics methods known in the art toidentify regions of secondary structure, e.g., one, two, or morestem-loop structures, or pseudoknots, and selecting those regions totarget with an inhibitory nucleic acid. For example, “gene walk” methodscan be used to optimize the inhibitory activity of the nucleic acid; forexample, a series of oligonucleotides of 10-30 nucleotides spanning thelength of a target RNA can be prepared, followed by testing foractivity. Optionally, gaps, e.g., of 5-10 nucleotides or more, can beleft between the target sequences to reduce the number ofoligonucleotides synthesized and tested. GC content is preferablybetween about 30-60%. Contiguous runs of three or more Gs or Cs shouldbe avoided where possible (for example, it may not be possible with veryshort (e.g., about 9-10 nt) oligonucleotides).

In some embodiments, the inhibitory nucleic acid molecules can bedesigned to target a specific region of the RNA sequence. For example, aspecific functional region can be targeted, e.g., a region comprising aknown RNA localization motif (i.e., a region complementary to the targetnucleic acid on which the RNA acts). Alternatively or in addition,highly conserved regions can be targeted, e.g., regions identified byaligning sequences from disparate species such as primate (e.g., human)and rodent (e.g., mouse) and looking for regions with high degrees ofidentity. Percent identity can be determined routinely using basic localalignment search tools (BLAST programs) (Altschul et al., J. Mol. Biol.,1990, 215, 403-410; Zhang and Madden, Genome Res., 1997, 7, 649-656),e.g., using the default parameters.

Pharmaceutical Compostions And Kits

Also provided herein are pharmaceutical compositions that include atleast one of any of the LSD1 inhibitors described and at least one ofany of the immunotherapies (e.g., at least one PD-1 and/or PD-L1inhibitor) described herein.

The pharmaceutical compositions can be formulated in any matter known inthe art. The pharmaceutical compositions are formulated to be compatiblewith their intended route of administration (e.g., intravenous,subcutaneous, intraperitoneal, rectal or oral). In some embodiments, thepharmaceutical compositions can include a pharmaceutically acceptablecarrier (e.g., phosphate buffered saline). Single or multipleadministrations of formulations can be given depending on for example:the dosage and frequency as required and tolerated by the patient. Thedosage, frequency and timing required to effectively treat a subject maybe influenced by the age of the subject, the general health of thesubject, the severity of the disease, previous treatments, and thepresence of comorbidities (e.g., diabetes). The formulation shouldprovide a sufficient quantity of active agent to effectively treat,prevent or ameliorate conditions, diseases or symptoms. Toxicity andtherapeutic efficacy of compositions can be determined usingconventional procedures in cell cultures, pre-clinical models (e.g.,mice, rats or monkeys), and humans. Data obtained from in vitro assaysand pre-clinical studies can be used to formulate the appropriate dosageof any composition described herein (e.g., any of the pharmaceuticalcompositions described herein).

Efficacy of any of the compositions described herein can be determinedusing methods known in the art, such as by the observation of theclinical signs of a cancer (e.g., tumor size, presence of metastasis).

Also provided herein are kits that include at least one of any of theLSD1 inhibitors described and at least one of any of theimmunotherapies, e.g., at least one PD-1 and/or PD-L1 inhibitor,described herein. In some instances, the kits can include instructionsfor performing any of the methods described herein. In some embodiments,the kits can include at least one dose of any of the pharmaceuticalcompositions described herein.

The disclosure is further described in the following examples, which donot limit the scope of the disclosure described in the claims.

EXAMPLES Example 1. Materials and Methods Cell Lines

MCF-7, T47D, B16, LLC and D4m cells were cultured in normal DMEMsupplemented with 10% FBS and 1% penicillin/streptomycin in 5% CO₂incubator at 37° C. All cell lines were cultured in 5% CO₂ incubator at37° C., and passaged every 2-3 days. One day before compound treatment,cells were seeded in 6-well or 12-well plates, and then were treatedwith 1, 2, or 5 μM GSK-LSD1, or DMSO as mock, in duplicates ortriplicates for 5-6 days, during which cells were passaged once andreplenished with fresh compound.

Mice

6-10-wk-old female mice were used for all experiments. WT C57BL/6 micewere purchased from The Jackson Laboratory. Prior to all experiments,purchased mice were allowed one week to acclimate to housing conditionsat the Harvard Medical School Animal Facility. For studies usingimmunodeficient mice, an in-house strain of WT mice was compared to anin-house strain of TCRα^(−/−) mice. The in-house strains of WT andTCRα^(−/−) were originally purchased from The Jackson Laboratory.Colonies for each strain of mice were maintained in the same animalfacility at Harvard Medical School. All experimental mice were housed inspecific pathogen-free conditions and used in accordance with animalcare guidelines from the Harvard Medical School Standing Committee onAnimals and the National Institutes of Health. Animal protocols wereapproved by the Harvard Medical School Standing Committee on Animals.

Gene Knockdown by shRNA and Rescue Assay

The shRNA oligos, with sequences for their respective target geneslisted in Table 2, were annealed and cloned into pLKO.1-Puromycin⁺(Puro) or pLKO.1-Blasticidin⁺ (Bsd) lentiviral vector. Lentiviruscarrying pLKO.1 plasmid was produced by co-transfecting HEK293T cellswith four helper plasmids (pHDM-VSV-G, pHDM-tat1b, pHDM-HgPM2, andpRC-CMVRaII), and by harvesting viral supernatant after 72 h by passingthrough a 0.45 μm filter. Collected lentivirus was used directly byinfecting cells with the addition of 8 μg/ml polybrene (Sigma-Aldrich,cat #H9268), or frozen at −80° C. for later use. Infected cells wereselected and expanded with puromycin (Gold Biotechnology, cat#P-600-500) at 1 μg/ml or blasticidin (Sigma-Aldrich, cat #15205) at 5μg/ml for 5 days before being used for subsequent assays.

For double KD, MCF-7 cells were first transduced with lentiviralpLKO-sh-Scramble-Bsd or pLKO-sh-LSD1-Bsd, and selected with blasticidinfor 3 days. Those cells were then transduced again with lentiviralpLKO-sh-GFP-Puro as control or pLKO-sh-Target-Puro, and selected withboth blasticidin and puromycin for 3-5 days to achieve double KD. Inthis context, pLKO-sh-Scramble-Bsd plus pLKO-sh-GFP-Puro was referred assh-C, and pLKO-sh-LSD1-Bsd plus pLKO-sh-GFP-Puro was referred assh-LSD1.

For LSD1 rescue assay, MCF-7 cells were first transduced with lentiviralpLKO-sh-Scramble-Bsd or pLKO-sh-LSD1-Bsd, and selected with blasticidinfor 3 days. Those cells were then transduced again with lentiviralpHAGE-CMV-Flag-HA-EV/LSD1/LSD1-K661A (puromycin and sh-LSD1 resistant),and selected with both blasticidin and puromycin for 5 days beforesubsequent analysis. In this context, pLKO-sh-Scramble-Bsd pluspHAGE-CMV-Flag-HA-EV was referred as sh-C, and pLKO-sh-LSD1-Bsd pluspHAGE-CMV-Flag-HA-EV was referred as sh-LSD1. For alternative rescuemethod, MCF-7 cells were first transduced with lentiviralpLKO-sh-Scramble-Bsd or pLKO-sh-LSD1-Bsd followed by blasticidinselection for 5 days before subsequent analysis.

TABLE 2 DNA oligonucleotide sequences shRNA Target Sequences SEQ ID NO:GFP GCAAGCTGACCCTGAAGTTCA SEQ ID NO: 7 T Scramble CCTAAGGTTAAGTCGCCCTCGSEQ ID NO: 8 Human LSD1 GCCTAGACATTAAACTGAATA SEQ ID NO: 9 Human TLR3CCTTACACATACTCAACCT SEQ ID NO: 10 Human MDA5 CCAACAAAGAAGCAGTGTATASEQ ID NO: 11 Human RIG-I AATTCATCAGAGATAGTCA SEQ ID NO: 12 Human MAVSGCATCTCTTCAATACCCTT SEQ ID NO: 13 #1 Human MAVS GGAGAGAATTCAGAGCAAGSEQ ID NO: 14 #2 Human cGAS ATCTATTCTCTAGCAACTTAA SEQ ID NO: 15Human STING GCATGGTCATATTACATCG SEQ ID NO: 16 Human AGO2GCACAGCCAGTAATCGAGTTT SEQ ID NO: 17 Human DICER AAGAATCAGCCTCGCAACAAASEQ ID NO: 18 #1 Human DICER TCTATTAGCACCTTGATGT SEQ ID NO: 19 #4Human TRBP2 GCTGCCTAGTATAGAGCAA SEQ ID NO: 20 #1 Human TRBP2TCTACGAAATTCAGTAGGA SEQ ID NO: 21 #2 Human TRBP2 GGATTCTCTACGAAATTCASEQ ID NO: 22 #4 Mouse LSD1 #1 CACAAGGAAAGCTAGAAGA SEQ ID NO: 23Mouse LSD1 #2 GGATGGGATTTGGCAACCTTA SEQ ID NO: 24 Mouse LSD1 #3AACTCCATGTCATCAGCTACT SEQ ID NO: 25 Mouse LSD1 #4 CGGCATCTACAAGAGGATAAASEQ ID NO: 26 CRISPR gRNA target Sequences SEQ ID NO: Mouse LSD1 #3ATATTCATCTTCTGAGAGGT SEQ ID NO: 27 (target location exon 2)Mouse LSD1 #4 TCTTCCTCAGGTGGGGCTTG SEQ ID NO: 28 (target locationexon 2) Mouse LSD1 #5 CCTGAGAGGTCATTCGGTCA SEQ ID NO: 29(target location exon 3) Mouse LSD1 #6 CCATGACCGAATGACCTCTCSEQ ID NO: 30 (target location exon 3) Mouse MDA5 GGCAGGGATTCAGGCACCATSEQ ID NO: 31 #4 (target location exon 4) RT-qPCR primer target SequenceSEQ ID NO: Human GAPDH AACGGGAAGCTTGTCATCAA SEQ ID NO: 32 forwardHuman GAPDH TGGACTCCACGACGTACTCA SEQ ID NO: 33 reverse Human LSD1GTGGACGAGTTGCCACATTTC SEQ ID NO: 34 forward Human LSD1TGACCACAGCCATAGGATTCC SEQ ID NO: 35 reverse Human HERV-EGGTGTCACTACTCAATACAC SEQ ID NO: 36 forward Human HERV-EGCAGCCTAGGTCTCTGG SEQ ID NO: 37 reverse Human HERV-F CCTCCAGTCACAACAACTCSEQ ID NO: 38 forward Human HERV-F TATTGAAGAAGGCGGCTGG SEQ ID NO: 39reverse Human HERV-K ATTGGCAACACCGTATTCTGC SEQ ID NO: 40 forward THuman HERV-K CAGTCAAAATATGGACGGATG SEQ ID NO: 41 reverse GT Human HML-2AAAGAACCAGCCACCAGG SEQ ID NO: 42 forward Human HML-2CAGTCTGAAAACTTTTCTCTA SEQ ID NO: 43 reverse Human ERVLATATCCTGCCTGGATGGGGT SEQ ID NO: 44 forward Human ERVLGAGCTTCTTAGTCCTCCTGTGT SEQ ID NO: 45 reverse Human Line1GCCAAGATGGCCGAATAGG SEQ ID NO: 46 forward Human Line1TGGCACTCCCTAGTGAGATGA SEQ ID NO: 47 reverse A Human AluYA5ACCATCCCGGCTAAAACGGTG SEQ ID NO: 48 forward A Human AluYA5GCGATCTCGGCTCACTG SEQ ID NO: 49 reverse Human IFN-αAATGACAGAATTCATGAAAGC SEQ ID NO: 50 forward GT Human IFN-αGGAGGTTGTCAGAGCAGA SEQ ID NO: 51 reverse Human IFN-ßGCCATCAGTCACTTAAACAGC SEQ ID NO: 52 forward Human IFN-ßGAAACTGAAGATCTCCTAGCC SEQ ID NO: 53 reverse T Human IL-28a/bTCCAGTCACGGTCAGCA SEQ ID NO: 54 forward Human IL-28 a/bCAGCCTCAGAGTGTTTCTTCT SEQ ID NO: 55 reverse Human OASLGCAGAAATTTCCAGGACCAC SEQ ID NO: 56 forward Human OASLCCCATCACGGTCACCATTG SEQ ID NO: 57 reverse Human ISG15 CCTTCAGCTCTGACACCSEQ ID NO: 58 forward Human ISG15 CGAACTCATCTTTGCCAGTAC SEQ ID NO: 59reverse A Human TLR3 TGGTTGGGCCACCTAGAAGTA SEQ ID NO: 60 forwardHuman TLR3 TCTCCATTCCTGGCCTGTG SEQ ID NO: 61 reverse Human MDA5CACTTCCTTCTGCCAAACTTG SEQ ID NO: 62 forward Human MDA5GAGCAACTTCTTTCAACCACA SEQ ID NO: 63 reverse G Human RIG-ICCAGCATTACTAGTCAGAAGG SEQ ID NO: 64 forward AA Human RIG-ICACAGTGCAATCTTGTCATCC SEQ ID NO: 65 reverse Human MAVSAGGAGACAGATGGAGACACA SEQ ID NO: 66 forward Human MAVS CAGAACTGGGCAGTACCCSEQ ID NO: 67 reverse Human cGAS TAACCCTGGCTTTGGAATCAA SEQ ID NO: 68forward AA Human cGAS TGGGTACAAGGTAAAATGGCT SEQ ID NO: 69 reverse TTHuman STING AGCATTACAACAACCTGCTAC SEQ ID NO: 70 forward G Human STINGGTTGGGGTCAGCCATACTCAG SEQ ID NO: 71 reverse Human AGO2CCGGCCTTCTCTCTGGAAAA SEQ ID NO: 72 forward Human AGO2GCCTTGTAAAACGCTGTTGCT SEQ ID NO: 73 reverse Mouse LSD1GTGGTGTTATGCTTTGACCGT SEQ ID NO: 74 forward Mouse LSD1GCTGCCAAAAATCCCTTTGAG SEQ ID NO: 75 reverse A MuERV-LTTTCTCAAGGCCCACCAATAG SEQ ID NO: 76 forward T MuERV-LGACACCTTTTTTAACTATGCG SEQ ID NO: 77 reverse AGCT Mouse MusDGATTGGTGGAAGTTTAGCTAG SEQ ID NO: 78 forward CAT Mouse MusDTAGCATTCTCATAAGCCAATT SEQ ID NO: 79 reverse GCAT Mouse IAP PolCTTGCCCTTAAAGGTCTAAAA SEQ ID NO: 80 forward GCA Mouse IAP PolGCGGTATAAGGTACAATTAAA SEQ ID NO: 81 reverse AGATATGG Mouse Line1TTTGGGACACAATGAAAGCA SEQ ID NO: 82 forward Mouse Line1CTGCCGTCTACTCCTCTTGG SEQ ID NO: 83 reverse Mouse IFN-a1CGGTGCTGAGCTACTGGC SEQ ID NO: 84 forward Mouse IFN-a1TTTGTACCAGGAGTGTCAAGG SEQ ID NO: 85 reverse Mouse IFN-bGGTGGAATGAGACTATTGTTG SEQ ID NO: 86 forward Mouse IFN-bAGGACATCTCCCACGTC SEQ ID NO: 87 reverse Mouse IL-28b AGCTGCAGGTCCAAGAGCGSEQ ID NO: 88 forward Mouse IL-28b GGTGGTCAGGGCTGAGTCATT SEQ ID NO: 89reverse Mouse ISG15 GGTGTCCGTGACTAACTCCAT SEQ ID NO: 90 forwardMouse ISG15 TGGAAAGGGTAAGACCGTCCT SEQ ID NO: 91 reverse Mouse OASLCAGGAGCTGTACGGCTTCC SEQ ID NO: 92 forward Mouse OASLCCTACCTTGAGTACCTTGAGC SEQ ID NO: 93 reverse AC Mouse TLR3GTGAGATACAACGTAGCTGAC SEQ ID NO: 94 forward TG Mouse TLR3TCCTGCATCCAAGATAGCAAG SEQ ID NO: 95 reverse T Mouse RIG-IAAGAGCCAGAGTGTCAGAATC SEQ ID NO: 96 forward T Mouse RIG-IAGCTCCAGTTGGTAATTTCTT SEQ ID NO: 97 reverse GG Mouse beta-actinGGCTGTATTCCCCTCCATCG SEQ ID NO: 98 forward Mouse beta-actinCCAGTTGGTAACAATGCCATG SEQ ID NO: 99 reverse T Mouse GAPDHTGACCTCAACTACATGGTCTA SEQ ID NO: 100 forward CA Mouse GAPDHCTTCCCATTCTCGGCCTTG SEQ ID NO: 101 reverse GFP-com GAACGGCATCAAGGTGAACTTSEQ ID NO: 102 forward GFPL reverse TAGCGTAATCTGGAACATCGT SEQ ID NO: 103ATGGGT GFP-let7 reverse GACGACCTCGAGTGAGGTAGT SEQ ID NO: 104 AGGTTGTATAGene/Strand- specific PCR primer target Sequence SEQ ID NO:Non-human Tag GCACACGACGACAGACGACG SEQ ID NO: 105 CAC HERV-E tag-TFGCACACGACGACAGACGACG SEQ ID NO: 106 CACCCAGAGTCAGGTGTCACT ACTCAATACACHERV-E tag-BR GCACACGACGACAGACGACG SEQ ID NO: 107 CACTACTGGAGCAACACGCAGCCTAGGTCTCTGG HERV-E TF GGTGTCACTACTCAATACAC SEQ ID NO: 108 HERV-E BRGCAGCCTAGGTCTCTGG SEQ ID NO: 109 HERV-K tag- GCACACGACGACAGACGACGSEQ ID NO: 110 RF(3) CACGGGAAGAATGTGTGGCCA ATAGTGCGGT HERV-K tag-GCACACGACGACAGACGACG SEQ ID NO: 111 BR(3) CACGGTAGAGATTCCTTTTTCTCCCCATTCCCAG HERV-K RF(3) GTGTGGCCAATAGTGCGGT SEQ ID NO: 112HERV-K BR(3) ATTCCTTTTTCTCCCCATTCCC SEQ ID NO: 113 AG Syn1-tag-TFGCACACGACGACAGACGACG SEQ ID NO: 114 CACATGGAGCCCAAGATGCAG TCCAAGASyn1-tag-BR GCACACGACGACAGACGACG SEQ ID NO: 115 CACCTAACTGCTTCCTGCTGAATTGGGGCGTA Syn1-TF ATGGAGCCCAAGATGCAG SEQ ID NO: 116 Syn1-BRCTAACTGCTTCCTGCTGAATT SEQ ID NO: 117 GGGGCGTAG Human beta-GCACACGACGACAGACGACG SEQ ID NO: 118 Actin-tag-TF CACGCTCGTCGTCGACAACGGCTCCGGCAT Human beta- GCACACGACGACAGACGACG SEQ ID NO: 119 Actin-tag-BRCACCAAACATGATCTGGGTCA TCTTCTC Human beta- GCTCGTCGTCGACAACGGCTCSEQ ID NO: 120 Actin-TF CGGCA Human beta- CAAACATGATCTGGGTCATCTSEQ ID NO: 121 Actin-BR TCTC

Gene Deletion by CRISPR/Cas9

The gRNA oligos, with sequences for their respective target genes listedin Table 2, were annealed and cloned into Lenti-CRISPR-v2-Puromycin⁺vector. To delete target genes, B16 cells were transiently transfectedwith Lenti-CRISPR-v2 plasmid carrying respective gRNA, and selected with1 μg/ml puromycin for 2 days. Cells were then transferred into freshmedium without puromycin and seeded at super-low density to allow colonyformation from single cell. Colonies were then picked up and expandedfor KO validation by immunoblot and by sequencing of target genomicregion. For double KO, LSD1 KO B16 cells (clone g5-4) were used fordeleting the second target gene as described above.

Generation of ERV Expression Construct and Transduction

Primers with sequences listed in table Si were used to get 2kb fragmentof HERV-K and 2kb fragment of HERV-E through PCR amplification withinsertion of stop codons at 5′ ends and additional 30 bp elongationprimers at 3′ ends. HERV-K and HERV-E fragments with reversecomplementary elongation primers at their 3′ ends were mixed, denatured,annealed and elongated, followed by PCR amplification to generate 4kbHERV-(K+E) fusion fragment, which was further cloned intopHAGE-CMV-Flag-HA lentiviral vector thus expressing sense transcript ofHERV-K and antisense transcript of HERV-E. Viral package andtransduction were performed at described above. MCF-7 cells transducedwith HERV-(K+E) were cultured for 48 hours without drug selection beforesubsequent analysis.

RNA Extraction and RT-qPCR

All reagents, buffers and containers used for RNA work were RNase-freegrade or treated with 0.1% v/v DEPC (Sigma-Aldrich cat #D5758) ifapplicable, to eliminate RNase contaminants in this section and otherrelevant sections. For total RNA extraction, cells in culture weredirectly lysed in TRIzol (Life Technologies, cat #15596018) after mediumremoval. RNA extraction was performed according to the manufacture'sinstructions. The extracted RNA was reversely transcribed into cDNAusing PrimeScript™ RT Reagent Kit (Clontech cat #RR037B) according tothe manufacturer's instructions, with following modifications: 2 μg ofRNA samples with the addition of primers were first denatured at 70° C.for 5 min and cooled down on ice before the addition of buffer andreverse transcriptase; incubation time (at 37° C.) was increased up to30 min. The obtained cDNA samples were diluted and used for real-timequantitative PCR (RT-qPCR). SYBR green (Roche, cat #06649416001) andgene specific primers with sequences listed in Table 2 were used for PCRamplification and detection on a LightCycler 480 system (Roche).

Strand-Specific PCR for Detection of Sense and Antisense ERV Transcripts

The strand-specific PCR method was adapted from (Chiappinelli et al.,2015) and performed with PrimeScript™ RT Reagent Kit (Clontech cat#RR037B) with modifications. In brief, gene- and strand-specific primers(GSP) were synthesized with an extra Tag sequence (listed in Table 2,which does not exist in human genome) at 5′-end to generate Tag-GSP (forexample, HERV-E Tag-BR for sense strand, Tag-TF for antisense strand),and were used for reverse transcription, following these steps: 1 μgtotal RNA in 6 μl H₂O was mixed with 1 μl Tag-GSP (10 μM stock),pre-heated at 65° C. for 5 min and cooled down on ice; then added 2 μlbuffer (5×), 0.5 μl reverse-transcriptase and 120 ng actinomycin D(Sigma-Aldrich, cat #A9415) in 0.5 μl H₂O to a total volume of 10 μl;incubated at 50° C. for 50 min for only first strand cDNA synthesis anddeactivated at 85° C. for 5 min; finally added 1 U RNase H (New EnglandBiolabs, cat #M0297S) and incubated at 37° C. for 20 min, followed byethanol precipitation for cDNA purification. The obtained cDNA was thenused for PCR amplification with paired primers: Tag-primer in pair withTF-primer for amplifying sense strand and Tag-primer in pair withBR-primer for amplifying antisense strand. The amplicons were visualizedon 1.5% agarose gels.

DsRNA Analysis by RNase Digestion and RT-qPCR

For dsRNA analysis by RNase A digestion, 5 μg total RNA extracted fromMCF-7 or B16 cells was dissolved in 46 μl H₂O and mixed well with 3.5 μlNaCl (5 M stock). Then 0.5 μl RNase A (10 mg/ml stock, Thermo FisherScientific, cat #EN0531) or H₂O as mock was added to a total volume of50 μl and mixed well, followed by incubation at room temperature for 10min. Afterwards, 1 ml TRIzol was directly added to the mixture toterminate digestion, followed by RNA extraction. The RNA transcripts ofselected retrotransposons were measured by RT-qPCR with GAPDH (Actb forB16 cells) as an internal control. The ratios of(retrotransposon/GAPDH)RN-A/(retrotransposon/GAPDH)_(mock) werecalculated as enrichment fold.

For dsRNA analysis by RNase T1 digestion, 2 μg total RNA extracted fromMCF-7 was dissolved in 16 μl H₂O and mixed well with 2 μl buffer (10×).Then 2 μl RNase T1 (1 U/μl stock, Thermo Fisher Scientific, cat #AM2283)or H₂O as mock was added to a total volume of 20 μl and mixed well,followed by incubation at 37° C. for 30 min. Afterwards, 1 ml TRIzol wasdirectly added to the mixture to terminate digestion, followed by RNAextraction and analysis as described above.

DsRNA Analysis by J2 Pulldown

Purified total RNA from control or LSD1 KD MCF-7 cells was used for J2pulldown assay. J2 antibody (Scicons, cat #10010200) and mouse IgGcontrol (Santa Cruz Biotechnology, cat #sc-2025) were first conjugated(1 μg per pulldown) to Protein G dynabeads (Life Technologies, cat#10008D), respectively. For each pulldown, 30 μg RNA was mixed with 500μl immunoprecipitation (IP) buffer (350 mM NaCl, 25 mM Tris pH7.4, 5 mMDTT and 0.5% NP-40), followed by the addition of 0.5 μl RNase A (10mg/ml stock, Thermo Fisher Scientific, cat #EN0531) and thorough mixing.The addition of RNase A was to reduce the overwhelming single-strandedRNA (ssRNA) and enrich dsRNA for J2 capture. Then, the whole mixture wasmixed with washed beads and rotated at 4° C. for 2 h. Afterwards, thebeads were washed with IP buffer and incubated in 50 μl Proteinase Kdigestion solution (1×TE, 100 mM NaCl, 1% SDS, and 1 μl Proteinase K (20mg/ml stock, Thermo Fisher Scientific, cat #AM2546)) at 45° C. for 20min. The elutes were directly added to 1 ml TRIzol for RNA purificationand RT-qPCR analysis as described above.

DsRNA Analysis by J2 Immunoblot

Purified total RNA from B16 cells was subjected to digestion with mock,RNase T1 (Thermo Fisher Scientific, cat #AM2283) and RNase III (ThermoFisher Scientific, cat #AM2290) in their respective buffers andaccording to the manufacturer's instructions, or RNase A (Thermo FisherScientific, cat #EN0531) under high salt condition (350 mM NaCl). Thedigestion was deactivated by the addition of TRIzol and RNA wassubsequently purified. Equal volume (2.5 μl) of purified RNA was dottedon Hybond N+ membrane (GE Healthcare, cat #RPN119B), dried andautocrosslinked in a UV stratalinker 2400 (Stratagene) two times. Themembrane was then blocked in 5% milk in PBS-T (0.1% Tween-20) for 30 minand probed with J2 antibody at 4° C. overnight. On the next day, themembrane was washed with PBS-T three times and probed with secondarygoat-anti-mouse HRP antibody (Millipore cat #AP124P) in 5% milk at roomtemperature for 1 h. The membrane was washed again with PBS-T threetimes and ECL was applied for film development. Afterwards, the membranewas stained with methylene blue solution (0.3% w/v methylene blue+30%v/v ethanol+70% v/v H₂O) to visualize RNA presence.

Protein Extraction and Immunoblot Analysis

Cells in culture were washed with ice-cold PBS twice to completelyremove residual medium. RIPA lysis buffer (150 mM NaCl, 1% NP-40, 0.5%sodium deoxycholate, 0.1% SDS and 50 mM Tris pH8) supplemented withprotease inhibitor (Roche, cat #0469313001) and phosphatase inhibitor(Roche, cat #04906837001) was directly added to cell layers and scrapedon ice. Cell lysates were transferred to small tubes and lysed on icefor 10 min before being cleared by top-speed centrifugation at 4° C.Protein concentrations in lysates were measured by Bio-Rad protein assay(Bio-Rad, #5000006) and adjusted equally between samples, followed bythe addition of SDS loading dye (5×) and boiled at 95° C. for 5 min.Equal volume and equal quantity of protein samples were subjected toSDS-PAGE and transferred to nitrocellulose membrane (Bio-Rad, cat#162-0097). The membrane was blocked in 5% milk at room temperature for1 h and incubated with appropriate antibodies at 4° C. overnight. On thenext day, the membrane was washed with PBS-T three times and incubatedwith appropriate secondary HRP antibodies in 5% milk at room temperaturefor 1 h. The membrane was washed again with PBS-T three times and ECLwas applied for film development.

ELISA

The ELISA assay was performed with a Human IFN Beta ELISA Kit (pbl assayscience, cat #41415-1) according to the manufacturer's instructions.

LSD1 Demethylase Assay

LSD1 demethylase assays were carried out with proteinsimmunoprecipitated by anti-HA magnetic beads from MCF-7 cells stablyexpressing FH-LSD1, FH-LSD1-K661A, FH-AGO2 and FH-AGO2-K726R In order toincrease basal methylation level on purified AGO2, MCF-7 cells stablyexpressing FH-AGO2 and FH-AGO2-K726R were treated with 2 μM GSK-LSD1 for24 hours before being used for IP purification. In a reaction of 50 μlvolume, immunoprecipitated LSD1 (˜2 μg, estimated by SDS-PAGE andcoomassie blue staining) and AGO2 (˜1 μg) were incubated indemethylation buffer (50 mM Tris pH 8.5, 50 mM KCl, 5 mM MgCl₂, 0.5%BSA, 5% glycerol, and complete EDTA-free protease inhibitors) on athermoshaker at 37° C. for 4 hours. The reaction was terminated byadding SDS loading dye (5×) and boiling at 95° C. for 5 min, followed bySDS-PAGE and immunoblot analysis. In each experiment, calf thymushistones were used as LSD1 demethylase substrate in parallel to checkthe activity of immunoprecipitated LSD1 by immunoblot analysis ofH3K4me2.

GFPL/GFP-Let-7 Dual Reporter Assay

The reporter assay for miRISC activity was performed as previouslydescribed (Qi et al., 2008). In brief, U2OS cell line stably expressingdual reporters, GFPL and GFP-let-7, was transduced with lentiviral shRNAagainst scramble, LSD1 or AGO2. Cells were selected with puromycin at 1μg/ml and G-418 (Research Products International, cat #G64500) at 200μg/ml for 4 days before subsequent analysis. The expression of GFPL andGFP was measured by immunoblot and RT-qPCR as described in the abovesections. Protein signals in immunoblot were quantified by ImageJsoftware according to the user manual. The ratios of GFPL over GFPprotein signals in different samples were calculated and the ratio incontrol shRNA sample was considered as 100% miRISC activity.

Cell Colony Formation Assay

B16 cells growing at 80% confluence were trypsinized and transferredinto fresh medium in single cell suspension. Cell numbers were countedand diluted appropriately for seeding to 6-well plates (500 cells perwell) or 12-well plates (200 cells per well). Cells were allowed to growfor 6 days, with fresh medium addition at day 3 without absorbing oldmedium, before staining with crystal violet solution (0.5% w/v crystalviolet powder, 80% v/v H₂O and 20% v/v methanol).

Mouse Tumor Models

Mice were anesthetized with Avertin (2.5%), shaved at the injectionsite, and then injected in the flank subcutaneously with 250,000-500,000B16-F10 tumor cells. Tumors were measured every 2-3 days once palpable(long diameter and short diameter) with a caliper. Tumor volume wasdetermined using the volume formula for an ellipsoid: 1/2×D×d² where Dis the longer diameter and d is the shorter diameter. Mice weresacrificed when tumors reached 2 cm³ or upon ulceration/bleeding.

For antibody treatments, mice were given 100 μg antibody i.p. at days14, 16, 18, and 20 post tumor injection using the following antibodies:anti-PD-1 (clone 29F.IA12) kindly provided by G. Freeman (Dana FarberCancer Institute, Boston, MA). Rat IgG2a isotype control antibody waspurchased from BioXCell (cat #BE0089). Prior to treatments mice wererandomized such that treatment groups had similar average tumor volumesprior to treatment initiation.

B16 Metastasis Assay

200K B16.F10 (scramble or LSD1 KO) were transferred intravenously viatail vein injection. Lungs were removed 14 days post injection and fixedovernight in Fekete's solution. Visible metastases were counted inblinded fashion by two investigators.

Tumor Infiltrating Leukocyte Flow Cytometry

Tumors were excised day 14 post injection and cut into 2 mm sized piecesin collagenase and DNase. Samples were dissociated with a Gentle MACS,incubated for 20 minutes at 37° C., dissociated with a Gentle MACSagain, and passed through a 70 μm filter. To enrich for leukocytessamples were spun through a Percoll gradient. Leukocytes were isolatedfrom the interface of the 40 and 70% Percoll gradient, stained, andanalyzed for fluorescent markers. For intracellular staining theEbioscience Foxp3 Fixation Permeabilization Kit was used. All antibodieswere purchased from Biolegend (CD45.2, CD11b, CD3, CD4, CD8b, Foxp3,Granzyme-B, Ki67, CD44, PD-L1, MHC-1).

Tumor Infiltrating Lymphocyte TCR Sequencing

T cells were enriched from tumors as above followed by sorting for CD8b⁺T cells. Genomic DNA was extracted using a DNeasy Blood & Tissue kit(Qiagen, cat #69506) and submitted to Adaptive Biotechnologies for mouseTCRB CDR3 survey sequencing. Data was analyzed using AdaptiveBiotechnologies' online analysis platform.

Directional RNA-Seq of MCF-7 Cells

Purified total RNA was quantified by Qubit (Invitrogen) and analyzed byAgilent Bioanalyzer to assess RNA integrity. 1 μg RNA (RIN>9) was usedto generate rRNA-depleted RNA with NEBNext® rRNA Depletion Kit (NewEngland Biolabs, cat #E6310S) according to the manufacturer'sinstructions. The rRNA-depleted RNA was subjected to Agilent Bioanalyzerto ensure the complete removal of rRNA, and then used to generatedirectional RNA library with NEBNext® Ultra™ II Directional RNA LibraryPrep Kit for Illumina® (New England Biolabs, cat #E7760L) and NEBNext®Multiplex Oligos for Illumina® (New England Biolabs, cat #E7335L)according to the manufacturer's instructions. Library concentrationswere quantified by Qubit (Invitrogen) and mixed equally for sequencingat HiSeq 2500 (Illumina) to generate 50 bp reads from paired-ends. Theraw data are deposited at the Gene Expression Omnibus (GEO) under thesubseries entry GSE105001.

ChIP-Seq of MCF-7 Cells

Cell nuclei were obtained by lysing whole cells in hypotonic buffer (10mM HEPES pH 7.9, 10 mM KCl, 1.5 mM MgCl₂, 0.34 M sucrose, 10% v/vglycerol, 1 mM DTT, and 0.1% v/v Triton X-100) supplemented withprotease inhibitor. After washing with PBS, nuclei were fixed in 1%formaldehyde for 10 min at room temperature, followed by quenching in125 mM glycine. Nuclei were then washed twice with ice-cold PBS, lysedin ChIP sonication buffer (50 mM HEPES pH7.9, 140 mM NaCl, 1 mM EDTA, 1%Triton X-100, 0.1% sodium deoxycholate, 0.2% SDS) supplemented withprotease inhibitor, and were subjected to sonication to obtain DNAfragments of 300-800 bp. Subsequent procedures were carried out byfollowing the Epigenesys protocol (www.epigenesys.eu/en/). The followingantibodies were used to ChIP: anti-LSD1 (Abcan, cat #ab17721),anti-H3K4mel (Abcam, cat #ab8895), anti-H3K4me2 (EMDMillipore, cat#07-030), and mouse IgG (Santa Cruz Biotechnology, cat #sc-2025).

ChIP-Seq libraries were prepared using NEBNext® Ultra™ DNA Library PrepKit for Illumina® (New England Biolabs, cat #E7370L) and NEBNext®Multiplex Oligos for Illumina® (New England Biolabs, cat #E7335L)according to the manufacture's instructions. Library concentrations werequantified by Qubit (Invitrogen) and mixed equally for sequencing atHiSeq 2500 (Illumina) to generate 50 bp reads from single-end. The rawdata are deposited at the Gene Expression Omnibus (GEO) under thesubseries entry GSE105001.

Statistic Analysis

Statistical analyses were performed using GraphPad Prism 6 software. ForRT-qPCR and protein quantification, data were shown as mean±s.d., andconsidered statistically significant with p values <0.05 by unpairedStudent's t test. For tumor growth, data were presented as mean±s.e.m.,and considered statistically significant with p values <0.05 by unpairedStudent's t test for comparing two groups or by two-way ANOVA formultiple comparisons. For comparing mouse survival curves, Log-rank(Mantel-Cox) test was used.

ChIP-Seq & RNA-Seq Analysis

For ChIP-seq and RNA-seq data, all statistical analysis andvisualization was performed with R (version 3.4.0) unless otherwisespecified. Student's t-test was used to determine whether significantshift in mean occurs for all comparisons unless otherwise specified.

ChIP-Seq Analysis

Raw reads were aligned to hg19 or mm⁹ using bwa (version 0.7.2-r351)(PMID: 22569178). The resulted sam files were converted to barn withsamtools (version 0.1.18 (r982:295) (PMID: 21245279). MACS2 (version2.0.10.20131216) (PMID: 18798982) was used to call peak on the barnfiles. BedGraph files containing signal per million reads produced fromMACS2 were converted to bigwig files with ucsctool kit (315). ChIP-seqsignals were first extracted with bwtool (version 1.0) (PMID: 24489365)from bigwig files and then visualized in R.

Repeat annotation was downloaded from UCSC for hg19 and mm⁹, only ERVswere used for downstream analysis. To select ERVs, ERV familiesoriginated in Homo Sapien or Mus Musculus were downloaded from Repbase(http://www.girinst.org/repbase/). A peak catalogue consisting allpossible peak intervals in ChIP-seq (histones and LSD1) was produced andERVs were filtered with this catalogue. ChIP-seq signals were extractedwith bwtool (version 1.0) (PMID: 24489365) from bigwig files and thenvisualized in R.

RNA-Seq Analysis

Raw reads were aligned to hg19 or mm⁹ using STAR (v2.4.2a) with theparameter “quantMode” set as “GeneCounts” to produce count table foreach gene. Differential gene analysis was performed on gene raw countsin R with edgeR package (v3.18.1) (PMID: 22287627) from bioconductor.Read count table was filtered so that genes with at least one countacross conditions were kept. The negative binomial generalizedlog-linear model was used in differential analysis. A FDR cut-off of0.05 was used to determine significantly differentially expressed genes.The R package gProfileR (v0.6.4, PMID: 27098042) was used to performgene enrichment analysis on differential genes. Geneset enrichmentanalysis (GSEA) was performed with R package clusterProfiler (v3.4.4,PMID: 22455463).

The function analyzeRepeats.pl from Homer (PMID: 20513432) software wasused to get raw counts for repeats from RNA-seq data. Differentialexpression for repeats was performed with edgeR the same way as forgenes.

TCGA Data Analysis

For the association of LSD1 expression with survival, patient vitalstatus (dead and alive) was used as surrogate end-point and patientdichotomized by LSD1 expression. The proportional hazards (PH)assumption was tested using the cox.zph function in the R Survivalpackage (v2.41-3) with 0.1 as cutoff. A log-rank test was used insteadif the PH assumption failed.

For analyzing LSD1 expression versus T cell infiltration in each tumor,the total expression of CD8A (log 2 counts per million) was used toassess the infiltration of cytotoxic T-lymphocytes, and correlationswere computed versus LSD1 expression.

For human SKCM data, patients were divided into tertiles based on LSD1expression, and then the second and third tertiles were combined intoone group (named LSD1-int/high) due to a lack of observable differencein survival curves between them.

For gene ontology, the online DAVID (david.ncifcrf.gov/summary.jsp) wasused to analyze the differentially expressed genes (>2 fold) betweenLSD1 KD and WT control under the category of GOTERM_BP_DIRECT. Foridentification of enriched gene sets, the two expression data sets werefurther utilized and Gene Set Enrichment Analysis (GSEA) was performedbased on these normalized data using GSEA tool (www.broad.mit.edu/gsea/)with C2. The raw and processed data of ChIP-Seq and RNA-Seq aredeposited at the Gene Expression Omnibus (GEO) under the subseries entryGSE105001.

Example 2. Lysine-Specific Demethylase 1A (LSD1) Represses ERVExpression and Antivirus Mimicry in Human Cancer Cells

To identify chromatin regulators that control tumor responses to hostimmunity, a curated screening with compounds targeting chromatin factorswas initiated. The screen was designed with two readouts: up-regulationof ERV transcripts and interferon activation, based on the followingrationale: (1) ERVs are known to be transcriptionally silenced byepigenetic mechanisms; (2) interferons regulate tumor responses to hostimmunity (Parker et al. (2016) Nature Reviews Cancer 16:131-144); (3) apotential correlation between ERV activity and tumor immunity has beensuggested (Rooney et al. (2015) Cell 160:48-61; Kassiotis and Stoye(2016) Nat Rev Immunology 16(4):207-219) and these two events may belinked by interferon activation (Chiappinelli et al. (2015) Cell162(5):974-986).

In this screen, an LSD1 catalytic inhibitor, GSK-LSD1, was shown tosignificantly induce the up-regulation of a few randomly selected ERVs,type I and type III interferons, as well as interferon-stimulated genes(ISGs) in MCF-7 cells (FIG. 1A). Of note, the PCR primers detectedoverall transcript levels of the corresponding ERV subfamilies, whichmay be transcribed from multiple genomic loc. To ascertain that this wascaused by a GSK-LSD1 on-target effect, shRNA-mediated LSD1 knockdown(KD) was performed (FIG. 1B), which yielded essentially the same results(FIGS. 1C-D). Furthermore, re-introduction of wild type (WT) LSD1 butnot catalytic inactive LSD1 (LSD1-K661A) back into LSD1 KD cells fullyrestored repression of four tested ERVs, as well as IFN-0 and IL-28activation (FIGS. 1E-G). These results demonstrated that demethylaseactivity of LSD1 is necessary for ERV repression, which is consistentwith the LSD1 inhibitor result (FIG. 1A). Neither DNMT proteinexpression nor global DNA methylation was affected by LSD1 inhibition(FIGS. 1H and 1I), suggesting a DNA methylation-independent pathway. Inaddition, the induction of ISGs such as ISG15 was also suppressed byLSD1 rescue (FIG. 1J). These observations were recapitulated in T47D,another breast cancer cell line (FIG. 1K), and 293T cells, a kidney cellline (FIG. 1L) suggesting that these effects were not limited to MCF-7cells and may be of broad significance in human cancer cells. Together,these results suggest that LSD1 may repress the expression of a group ofERVs and regulate interferon activation in human cancer cells.

Next, transcriptomic analysis was carried out to comprehensively explorehow LSD1 regulates ERV expression and interferon activation. Asignificant impact of LSD1 inhibition on gene expression in MCF-7 cells(FIG. 1M). Gene ontology (GO) enrichment analysis of thesedifferentially expressed genes revealed that the up-regulated genes weresignificantly enriched in GO terms related to type I interferon responseand antiviral response (FIG. 1N), whereas the down-regulated genesseemed to be enriched in GO terms related to neuronal development (FIG.1O). GSEA analysis confirmed the remarkable enrichment in type Iinterferon and antiviral responsive pathways in LSD1 KD cells comparedto WT control (FIG. 2A). However, almost none of the up-regulatedinterferon/antiviral responsive genes (FDR<0.05 and log 2(FC)>0, 125 intotal) appeared to be direct targets of LSD1, as ChIP-seq analysisfailed to identify LSD1 at their promoters (Table 3 and FIG. 2B) Anexample of such genes indirectly regulated by LSD1 at their promotersand an example of LSD1 direct target genes were shown in FIGS. 2C and2D.

TABLE 3 List of up-regulated interferon/antiviral responsive genes inLSD1 KD cells compared to WT control, related to FIG. 2. No LSD1-boundLSD1-bound CD274, IFIT3, IFI6, HERC5, XAF1, DDX60, IFIT1, OAS2, OAS3,OASL, IFIH1, IFI35, DHX58, IFI44L, EPSTI1, APOL6, GBP4, TMEM106A, IFI44,IFIT5, MX1, EIF2AK2, RSAD2, APOBEC3G, APOL1, TNFSF10, LAMP3, TDRD7,PARP12, SAMD9L, MB21D1, RARRES3, STAT1, KIF5C, TRIM22, IRF7, CXCL10,CXCL11, SAMHD1, PLSCR1, PDZD2, IFI27, PSMB9, IFIT2, TLR3, ISG20, IL15BST2, SP110, STX11, UBE2L6, GBP2, GBP1, OAS1, GMPR, TRIM21, MX2, SP100,PSMB8, NMI, ISG15, HLA-F, TAP1, TYMP

Nevertheless, those genes are, by and large, downstream ISGs andtherefore are unlikely to be directly responsible for the up-regulationof no-LSD1-bound genes and interferon pathway activation. A main mode ofinterferon/antiviral responses by LSD1 inhibition is speculated to bethrough activation of an upstream event, such as ERV transcriptexpression. The expression of repetitive elements in RNA-seq data wasthen analyzed. Many of the repetitive elements were up-regulated by LSD1inhibition (FIG. 2E), including a number of ERVs (either sense oranti-sense transcripts) were significantly increased in LSD1 KD cells(FIG. 2F). Furthermore, many ERVs appeared to be direct targets of LSD1as they were bound by LSD1 and showed elevated H3K4me2 levels upon LSD1KD (FIGS. 2G and 2H). (HERV-E is shown as an example in FIG. 2I).Importantly, a number of up-regulated retrotransposons, including ERVs,LTRs and LINEs, were expressed in both sense and antisense directionswith overlapping sequences potentially allowing for the formation ofdouble stranded RNAs (FIG. 2J). This observation was confirmed byanalyzing a number of selected ERVs using strand-specific PCR (FIG. 2K).Thus, these findings demonstrated that LSD1 is important fortranscriptionally silencing ERVs, consistent with a previous reportsuggesting that LSD1 regulates the expression of repetitive elements inmouse embryonic stem cells (mESCs) (Macfarlan et al. (2011) Genes Dev25(6): 94-607).

To determine whether ERV transcript up-regulated caused by LSD1inhibition was a causal factor for the induction of IFN/antiviralresponsive genes, an engineered 4kb ERV fragment was ectopicallyexpressed without protein coding capacity, derived from HERV-K andHERV-E. Its RNA overexpression readily caused the induction of IFNs andISGs in MCF7 cells (FIGS. 2L and 2M), which demonstrated the sufficiencyof ERV up-regulation in triggering IFN activation.

Example 3. TLR3 and MDA5 Sense dsRNA Accumulation Caused by LSD1Abrogation, which Triggers Interferon Activation

To determine whether ERV up-regulation as well as other retrotransposonsin both sense and antisense directions in LSD1 KD cells contribute tothe generation of dsRNAs, which may then trigger interferon activation,RNases and a dsRNA-specific antibody (J2) were used (White et al. (2014)Nat Struct Mol Biol 21(6): 552-559) to measure the presence of dsRNA.RNase A (under high salt condition) or RNase T1 cleaves single stranded(ss) RNA and preserves dsRNA (Roulois (2015) Cell 162: 961-973). Bydigesting total RNA isolated from control and LSD1 KD cells, and bynormalizing to undigested RNA, the relative dsRNA enrichment wascalculated in the presence and absence of LSD1. dsRNA enrichment for anumber of ERVs as well as a few other retrotransposons was much higherin LSD1 KD samples as compared to control samples (FIGS. 2N and 2O).Using the dsRNA-specific J2 antibody, more transcripts of selectedretrotransposons were captured in LSD1 KD samples (FIG. 2O). Theseresults provide evidence for the elevation of intracellular dsRNAlevels, which are elevated as a result of LSD1 inhibition. IntracellulardsRNAs are recognized by pattern recognition receptors, TLR3, MDA5 andRIG-I, which are involved in subsequent activation of the interferonpathways (Takeuchi and Akira (201) Cell 140:805-820). In LSD1 KD cells,all three dsRNA sensors were among the up-regulated genes identified byRNA-seq (FIG. 2P). Furthermore, all three sensors were inducedconsiderably at the protein level in LSD1 KD cells as well (FIG. 2Q),implying that those sensors might be responsible for detectingintracellular dsRNA accumulation in the absence of LSD1. To identifywhich sensor was essential for recognizing dsRNAs to elicit cellularresponses, expression of individual sensors were inhibited byshRNA-mediated knockdown in LSD1 KD cells, and the impact of knockdownon interferon activation was assessed. Each shRNA efficiently knockeddown the expression of its target sensor (FIG. 3A). Importantly,abrogation of TLR3 and MDA5, but not RIG-I, significantly diminished theinduction of interferon-0, IL-28 as well as ISGs without altering ERVexpression level (FIGS. 3B-D and FIG. 4D). In addition, the abrogationof MAVS, which is a downstream adaptor of the MDA5 pathway, also blockedinterferon activation in LSD1 KD cells (FIGS. 3E and 4E). As a furthercontrol, two key molecules, cGAS and STING, were knocked down in thecytoplasmic DNA sensing pathway (Chen et al. (2016) Nat Immunol 17(10):1142-1149) and showed that cytoplasmic DNA is unlikely the trigger ofinterferon responses in LSD1 KD cells (FIGS. 3G and 3H). Therefore,dsRNA recognition by TLR3 and MDA5 was essential for IFN activation uponLSD1 inhibition, consistent with the observation that the up-regulatedIFN/antiviral responsive genes were indirect targets of LSD1 (FIG. 2B).

Previous studies of these sensors (Takeuchi and Akira (201) Cell140:805-820) suggest that TLR3 and MDA5 recognize dsRNAs that are atleast 40 bp in length or longer, respectively (see, e.g., Liu et al.(2008) Science 320(5874):379-381; Kato et al. (2006) Nature441(7089):101-105, and Kato et al. (2008) J Exp Med 205(7): 1601-1610),whereas RIG-I prefers ssRNA or short dsRNA with 5′ triphosphate ends(see, e.g., Pichlmair et al. (2006) Science 314(5801): 997-1001, Homunget al. (2006) Science 314: 994-997; and Kato et al. (2008) J Exp Med205(7): 1601-1610). The involvement of TLR3 and MDA5 in response todsRNA stress is consistent with the directional RNA-seq analysissuggesting that those enriched dsRNAs are sufficiently long to berecognized by the dsRNA sensors TLR3 and MDA5.

Example 4. Decreased RISC Activity Due to Loss of LSD1 ReinforcesIntracellular dsRNA Stress and Promotes IFN Activation

Double stranded RNAs derived from ERV transcripts can go on to triggerinterferon responses or be processed by the RISC complex to generateendogenous small interfering RNA (endo-siRNA) and RNA interference (see,e.g., Watanabe et al. (2008) Nature 453(7194): 539-543; Tam et al.(2008) Nature 453(7194): 534-538; and Okamura and Lai (2008) Nat Rev MolCell Biol 9(9): 673-678). Thus the steady state of dsRNA pool isdetermined not only by ERV transcription but also the action of the RISCcomplex. Next, it was determined whether LSD1 might also regulate theRISC complex to influence the steady state of dsRNA pool. LSD1 KD led toreduced protein expression of key components (DICER, AGO2 and TRBP2) ofRISC (FIG. 4A). The regulation of RISC is dependent on LSD1 catalyticactivity, as re-introduction of WT LSD1 but not LSD1-K661A back intoLSD1 KD cells restored the protein expression of DICER, AGO2 and TRBP2(FIG. 4A). In contrast, an obvious impact of LSD1 on the expression ofDrosha, a crucial enzyme for miRNA biogenesis was not observed (FIG.4B). Consistent with the above expression analysis, LSD1 KD alsoresulted in an elevated expression of a GFP reporter (FIGS. 4C, 4F and4G), whose expression was under the control of let-7 miRISC activity (Qiet al. (2008) Nature 455(7211): 421-424). This finding suggests thatRISC may also be involved in dsRNA stress and interferon responses.Indeed, when AGO2 expression was inhibited by shRNA, an increase wasobserved in dsRNA abundance derived from a few retrotransposons tested(FIG. 4H), leading to the induction of interferon-β and IL-28 as well asISGs (FIGS. 4I and 4J). Similarly, inhibition of either DICER or TRBP2also activated the interferon pathway (FIGS. 4K-4N). Therefore,disruption of the RISC complex, which perturbs intracellular dsRNAhomeostasis, is sufficient to elicit IFN activation. To confirm thatRISC complex is necessary for LSD1 inhibition-stimulated IFN activation,the reduction of RISC was compensated for by overexpressing AGO2 in LSD1KD cells. AGO2 overexpression significantly diminished dsRNAaccumulation caused by LSD1 inhibition, leading to a reduction in IFNactivation (FIGS. 4O-4Q). Collectively, these findings suggest that, inaddition to regulating ERV transcription, LSD1 also regulates theexpression of RISC components and consequently RISC activity. Bothactions of LSD1 contribute to its suppression of dsRNA accumulation.

Example 5. LSD1 Regulates AGO2 Protein Demethylation and Stability

In order to understand the mechanism by which LSD1 regulates theexpression of RISC components, it was determined whether LSD1inhibition-induced dsRNA stress, which was previously reported todecreases DICER protein expression (Wiesen and Tomasi, 2009), wasinvolved. To this end, dsRNA stress was released by blocking itsrecognition by dsRNA sensors; TLR3 KD fully restored the protein levelof DICER but not AGO2 or TRBP2 in LSD1 KD cells (FIG. 5A). This resultconfirmed that the regulation of DICER expression by LSD1 was indirect,through dsRNA stress, while it also suggested that the regulation ofAGO2 and TRBP2 expression was likely independent of dsRNA stress. Giventhat AGO2 is the central component responsible for RNA cleavage, therole of AGO2 was investigated in greater detail. No alterations in AGO2RNA levels were detected upon LSD1 KD (FIG. 5B), suggesting theregulation occurs at post-transcriptional level. Indeed, in acyclohexamide (CHX) chase assay, a substantial decrease in AGO2 proteinhalf-life was detected when LSD1 was inhibited (FIGS. 5C and 5D),implicating a regulatory role of LSD1 in AGO2 protein stability.Interestingly, LSD1 was found to physically interacted with RISC complexas shown by co-immunoprecipitation assays using whole cell lysate (WCL)of MCF-7 cells stably expressing FH-AGO2 or FH-TRBP2 (FIGS. 5E and 5F).In addition, this physical interaction likely occurred in the nucleus,because a portion of RISC components was detected in the nuclearfraction and LSD1 is exclusively localized in the nucleus (FIG. 5G). Asfurther evidence, reciprocal co-immunoprecipitation with WCL or nuclearextract (NE) of MCF-7 cells stably expressing FH-LSD1 was performed, anda much stronger interaction between LSD1 and AGO2 in NE was detectedcompared with that in WCL (FIG. 3H).

To investigate whether LSD1 regulates AGO2 stability by controlling AGO2methylation, overexpressed FH-AGO2 from MCF-7 cells with or without LSD1inhibition were purified and used for mass spectrometry analysis. Alysine residue at position 726 (K726) was consistently mono-methylatedwhen LSD1 was inhibited either by shRNA-mediated KD or by GSK-LSD1 (FIG.5I). To validate this finding, an antibody was raised thatpreferentially recognized AGO2 peptides mono-methylated at K726(K726mel) compared with un-methylated or di-methylated peptides (FIG.5J). Furthermore, this antibody detected increased K726mel onectopically expressed AGO2 when LSD1 was inhibited, which can beabrogated by substituting K726 with arginine (K726R) or alanine (K726A)(FIGS. 5K and 5L). Importantly, this methyl specific antibody alsodetected more mono-methylation at K726 on endogenous AGO2 upon LSD1inhibition (FIG. 5M), suggesting that LSD1 regulates AGO2 demethylationin vivo. To gain more insights into this regulation, an in vitrodemethylation assay was performed using immunoprecipitated FH-LSD1,FH-LSD1-K661A (catalytically compromised LSD1), FH-AGO2 andFH-AGO2-K726R proteins purified from mammalian cells. FH-LSD1 but notFH-LSD1-K661A decreased K726mel level on FH-AGO2, but had no observableeffects on FH-AGO2-K726R (FIGS. 5N and 5O). Together, these results showthat LSD1 regulates AGO2 demethylation at K726 in vivo, most likelythrough its catalytic activity against AGO2.

To ascertain that K726 demethylation is responsible for sustaining AGO2stability, its methylation was blocked by a substitution of lysine forarginine, and observed an increased stability for AGO2-K726R compared towild-type AGO2 under physiological condition as well as in response toLSD1 inhibition (FIG. 5P). Taken together, LSD1 modulates AGO2 stabilityby regulating AGO2 demethylation at K726, which is required for basalRISC activity that critically controls intracellular dsRNA homeostasis.Inhibition of LSD1 disrupts the above pathway, in addition to causingERV up-regulation, leading to dsRNA stress and IFN activation in humancancer cells.

Example 6. LSD1 Abrogation-Induced dsRNA Stress Suppresses Tumor CellGrowth In Vitro

To address the biological consequence of LSD1 inhibition-induced dsRNAstress, and in particular, whether dsRNA stress-triggered cellularresponses can be harnessed for anti-tumor immunity, C57BL/6 syngeneicmouse models were used. First, it was determined whether the previousobservations made in human cells could be recapitulated in mouse cells.Lewis lung carcinoma (LLC), D4.m3A cells and B16 melanoma cells are allmouse tumor cell lines on the C57BL/6 genetic background with poorimmunogenicity (Lechner et al. (2013) J Immunother 36(9): 477-489). LSD1inhibition by Clustered Regularly Short Palindromic Repeats(CRISPR)/Cas9-mediated gene deletion resulted in upregulation ofretrotransposons and activation of IFN pathways in those lines (FIGS.6A-6D and 7A-7F), which recapitulated the findings in human cellsdescribed in Examples 2-5. In addition, dsRNA accumulation was observedin response to LSD1 loss (FIGS. 6E-6G). Taken together, the presentresults showed that LSD1 restrained intracellular dsRNA stress andinterferon activation in both human and mouse cancer cells. LSD1inhibition either by shRNA-mediated KD or by CRISPR/Cas9-mediated KOresulted in compromised growth of B16 cells in vitro (FIGS. 6H-6K),consistent with what has been reported previously for LSD1 in other celllines (see, e.g., Zhang et al. (2013) Cell Rep 5(2): 445-457; Harris etal. (2012) Cancer Cell 21(4): 473-487; and Mohammad et al. (2015) CancerCell 28(1): 57-69). To determine whether the growth phenotype is due toLSD1 abrogation-induced dsRNA stress, the dsRNA sensor MDA5 or TLR3 wasdeleted in LSD1 KO B16 cells (FIGS. 6L and 6M). The deletion of MDA5significantly, albeit partially, rescued the growth defect of LSD1 KOB16 cells (FIGS. 6N and 6O), suggesting that the growth defect was inpart due to the dsRNA stress induced by LSD1 deletion. At the molecularlevel, the induction of interferons and ISGs, but not dsRNA abundance,was significantly diminished in the LSD1/MDA5 double knockout (DKO)cells (FIGS. 6P and 6Q). As a control, deletion of MDA5 alone hadminimal effects on B16 cell growth and interferon activation (FIGS.8A-C). No apparent rescue was observed by TLR3 genetic deletion (FIG.8D), which could be explained by the observation that TLR3 was not orminimally expressed in B16 cells (data shown). Therefore, similar tohuman cells, removal of dsRNA sensors blocks downstream interferonactivation triggered by dsRNA stress caused by LSD1 loss.

A similar rescue effect on cell growth by blocking IFN pathway in LSD1KO B16 cells. Indeed, the deletion of IFNAR1, a crucial subunit for type1 IFN receptor, also diminished IFN activation and partially restoredcell growth (FIGS. 8E-J), in line with the suppressive effect of type 1IFN on cell growth. In addition, IFN-β deletion also displayed asimilar, albeit milder, rescue effect (FIGS. 8K-L). LSD1-abrogation inmouse cancer cells causes dsRNA stress and subsequent IFN activation,leading to cell growth inhibition in vitro.

Example 7. LSD1 Abrogation-Induced dsRNA Stress Triggers Anti-Tumor TCell Immunity In Vivo

The role of LSD1 in basic cancer biology has been previously reported,which includes sustaining cancer stem cell self-renewal and suppressingdifferentiation, promoting cell proliferation, enhancing anepithelial-to-mesenchymal transition (EMT) as well as modulatingmetastasis (reviewed in Hosseini and Minucci, 2017). However, thosestudies used either in vitro cell culture systems or transplanted humancancer cells into immuno-deficient, in which the role of LSD1 inregulating tumor response to host immunity was not possibly to beexplored.

To determine whether LSD1 deletion-induced dsRNA stress and interferonactivation might trigger anti-tumor immunity in vivo. C57BL/6 WT micewere subcutaneously inoculated with B16 cells. The deletion of LSD1 inB16 cells significantly inhibited tumor growth in vivo (FIGS. 9A and9B), in agreement with the previous in vitro observations (FIGS. 6H-K).To distinguish the role of LSD1 in regulating tumor autonomous growthversus host anti-tumor immunity, both immunocompetent (WT) andimmunodeficient, T-cell receptor a (TCRα) KO, mice were used forsubcutaneous tumor growth assays. Although LSD1 deletion inhibited B16tumor growth in WT mice, there was no growth difference between LSD1 KOand control B16 tumors in the TCRα-deficient mice (FIGS. 9C and 9D).This result indicated that LSD1 inhibition in tumor cells elicits potentanti-tumor T cell immunity in vivo, rather than affecting tumorautonomous growth, to restrain tumor burden. The reason for the loss ofautonomous growth defects of LSD1 KO cells in vivo is not known at thepresent time, however, this could be due to the possibility that hostsomatic cells, such as stromal cells in the tumor microenvironment,foster cell proliferation and tumor growth by secreting growth-promotingfactors that may substitute the need for LSD1 in cell proliferation.

To confirm that host anti-tumor T cell immunity was boosted bytumor-intrinsic dsRNA stress as elucidated above, tumor growth of LSD1KO and LSD1/MDA5 DKO B16 cells was compared in immunocompetent mice.Deletion of MDA5 was sufficient to diminish LSD1 inhibition-elicitedanti-tumor immunity, evidenced by the finding that LSD1/MDA5 DKO tumorsdisplayed similar growth ability as control tumors targeted withscrambled gRNA or MDA5 single KO tumors (FIGS. 9E and 9F). To furtherexamine whether MDA5-associated type 1 IFN response is essential forLSD1 inhibition-elicited anti-tumor immunity, IFN-β production wasabrogated in LSD1 KO B16 tumors (FIGS. 6L and 8L), which completelyreverse growth inhibition to a level comparable to that of the controlof IFN-β single KO tumors in the immunocompetent mice (FIG. 9G). Inaddition to controlling tumor growth, LSD1 inhibition resulted in amarked reduction in B16 tumor metastasis (FIGS. 9H and 9I). Thus, LSD1inhibition-caused dsRNA stress and resultant IFN response sensitizetumors to T cell immunity, likely by increasing tumor immunogenicity.

This finding is consistent with previous reports demonstrating that LSD1promotes cell proliferation in cell culture and in mouse xenograftmodels (Zhang et al. (2013) Cell Rep 5(2): 445-457; and Mohammad et al.(2015) Cancer Cell 28(1): 57-69). However, since these studiestransplanted human tumor cells into immunodeficient mice, the potentialimpact from tumor microenvironments, in particular immune cells, was nottaken into consideration. When syngeneic immunodeficient andimmunocompetent mice were used in parallel, LSD1 was found not to berequired for B16 tumor autonomous growth in vivo, revealing that LSD1inhibition mainly elicits a potent anti-tumor adaptive immunity, whichdrastically reduces tumor growth.

Example 8. LSD1 Inhibition Manifests Tumor Immunogenicity and IncreasesT Cell Infiltration

To further elucidate the mechanism connecting LSD1 inhibition toenhanced anti-tumor T cell immunity, the impact of tumor cell-intrinsicLSD1 on T cell activity in the tumor microenvironment was determined. Byanalyzing tumor-infiltrating lymphocytes (TILs) in transplanted B16tumors, LSD1 ablation in tumor cells resulted in a significant increasein CD4⁺ and CD8⁺ T cell infiltration, indicating a stronger ability toinduce T cell immunity (FIG. 10A). Importantly, the increase in T cellinfiltration was diminished when MDA5 was concurrently ablated (FIG.10A). In contrast, no significant alteration in T cell populations wasdetected in draining lymph nodes (dLNs) of B16 tumor-bearing mice (FIG.10B), suggesting the impact on T cells by tumor cell LSD1 ablation isrestricted to tumor sites. To assess the functional activity of CD8⁺TILs, the expression of a proliferation marker, Ki-67, and a cytotoxicfactor, Granzyme-B (GzmB) were detected, but neither showed a noticeablealteration when LSD1 was deleted in B16 tumor cells (FIG. 10C). Thus, inconsideration of the aforementioned tumor growth inhibition (FIGS.9A-I), these results suggest that increased T cell infiltration mediatedby dsRNA recognition pathway imparts potent anti-tumor immunity toLSD1-null tumors.

To investigate whether the increased T cell infiltration is associatedwith increased TCR repertoire diversity of CD8⁺ TILs in LSD1 KO B16tumors, the clonality and entropy of these T cells was analyzed by Tcell receptor (TCR) sequencing, but no significant changes were foundcompared to their counterparts in WT tumors (FIG. 10D). Thus, theincreased T cell infiltration in LSD1 KO tumors is not due to anunlikely alteration in tumor antigenicity.

To investigate tumor cell characteristics, which are associated withtumor response to T cell immunity and critically regulated by LSD1inhibition-induced dsRNA stress, GFP-labeled B16 lines were created tofacilitate the accurate isolation of tumor cells from in vivotransplanted tumors, and then these ex vivo tumor cells were used fortranscriptomic analysis. LSD1 deletion significantly altered geneexpression profile in B16 tumor cells in vivo, which appeared to besuppressed when MDA5 was simultaneously deleted (FIGS. 10E and 10F).Consistent with the in vitro results with human cancer cells, LSD1ablation also led to upregulation of ERVs by regulating theirtranscription in B16 tumor cells in vivo (FIGS. 10G-I), suggesting theaforementioned mechanism is conserved in vivo.

Genes whose expression was selectively up-regulated (FDR <0.05 and log2(FC) >1) in LSD1 KO tumor cells compared with control tumor cells werefiltered out for GO analysis. This analysis showed that immuneresponse-related biological processes, including innate immune response,response to IFN-β, defense response to virus and MHC protein complex,were ranked among the top 10 GO terms in LSD1 KO tumor cells (FIG. 10J),providing evidence for the increased tumor immunogenicity. The GO termresponse to IFN-γ was also significantly enriched (FIG. 10J), implyingan increased response of LSD1 KO tumor cells to T cell killing. Inaddition, genes associated with inflammatory response were also enrichedin LSD1 KO tumor cells as analyzed by GSEA (FIG. 10K). Importantly, theinduced expression of genes associated with the top 10 GO terms wassignificantly diminished by simultaneous MDA5 deletion in LSD1 KO cells(FIG. 10L), suggesting a critical role of dsRNA recognition pathway inmediating tumor immunogenicity. Of note, there was no apparentalteration by LSD1 deletion of cell proliferation pathways by GSEA (FIG.10M), which further supported the notion that autonomous growth of B16tumor cells in syngeneic mice is likely independent of LSD1 status.

In order to validate the findings from RNA-seq, it was determinedwhether antigen presentation on tumor cell surface restricted by MHC-1,whose alteration enables immune escape and is commonly found in solidtumors, is affected by LSD1. In the RNA-seq analysis, most MHC-1 codinggenes were upregulated in LSD1 KO B16 cells, among which the inductionof H2-D1 and H2-K1, encoding classical class 1 antigens, was largelydependent on MDA5 pathway (FIG. 10N). Consistently, in flow cytometricanalysis of GFP-labeled B16 cells isolated from in vivo transplantedtumors, LSD1 deletion caused a marked induction of MHC-1 expression ontumor cell surface, which was completely abrogated by concurrentdeletion of MDA5 (FIG. 10O). Altogether, these results show that LSD1inhibition through the dsRNA recognition pathway manifests tumorimmunogenicity, associated with increased T cell infiltration.

To examine whether the enhanced tumor immunogenicity by LSD1 inhibitionis a generalizable mechanism, another “cold” tumor model, D4m melanoma,in which LSD1 ablation also caused increased dsRNA levels and IFNactivation, was used (FIGS. 6C and 6G). In syngeneic immunocompetentmice, LSD1 KO D4m tumors displayed slower growth than wild type controltumors (FIGS. 10P and 10Q). Consistently and critically, increased Tcell infiltration in LSD1 KO tumors and elevated MHC-1 expression on thesurface of LSD1 KO tumor cells was found compared with control tumors(FIGS. 10R and 10S), indicating enhanced T cell immunity. Thus, theseresults suggested that the enhanced tumor immunogenicity by LSD1inhibition was not limited to B16 tumor model and are of broadersignificance.

Notably, RNA-seq and flow cytometry identified up-regulation of PD-L1expression in the B16 tumor cells in vivo, which is independent of MDA5(FIGS. 10T and 10U). It is possible that PD-L1 induction may suppressthe functional activity of CD8⁺ TILs (Juneja et al., 2017), thuscompromising the anti-tumor effect of increased TILs caused by LSD1inhibition. In summary, our results reveal a critical impact of tumorcell-intrinsic LSD1 on modulating tumor response to T cell immunity.

Example 9. LSD1 Inhibition Overcomes Tumor Resistance to PD-1 Blockade

In cancer patients, the presence of CD8⁺ TILs that are suppressed byPD-L1 predicts the responsiveness to PD-(L)1 blockade (see, e.g., Herbstet al. (2014) Nature 515(7528): 563-567; and Tumeh et al. (2014) Nature515(7528): 568-571). B16 tumors have high expression of PD-L1 expressionbut poor immunogenicity, and are known to be non-responsive toPD-1/PD-L1 blockade in the absence of vaccination (see, e.g., Chen etal. (2015) Cancer Immunol Res 3(2): 149-160; Kleffel et al. (2015) Cell162(6): 1242-1256; and Juneja et al. (2017) J Exp Med 214(4): 895-904).Given that LSD1 inhibition elicits anti-tumor immunity, it wasdetermined whether LSD1 inhibition would sensitize B16 tumors toPD-1/PD-L1 blockade. Consistent with previous reports (see, e.g., Chenet al. (2015) Cancer Immunol Res 3(2): 149-160; Kleffel et al. (2015)Cell 162(6): 1242-1256; and Juneja et al. (2017) J Exp Med 214(4):895-904), PD-1 blockade alone had no overt effects on wild type B16tumor growth (FIGS. 11A and 11B). Strikingly, PD-1 blockade showed adramatic effect on controlling LSD1 KO B16 tumors (FIGS. 11A and 11B).Furthermore, this responsiveness to PD-1 blockade doesn't rely on tumorsize, because re-scheduled anti-PD-1 administration when tumor sizesreached a set volume also had a profound effect on controlling growth ofLSD1 KO tumors but not WT tumors (FIGS. 11C and 11D). Moreover, thisprofound delay in tumor growth was achieved with a late initiation ofPD-1 blockade as well as a low dose of blocking antibody. These resultsdemonstrated a strong synergy between LSD1 inhibition and PD-1 blockadein controlling tumor growth and suggest that targeting LSD1 bypasses theneed for vaccination to obtain PD-1 blockade responsiveness in the B16tumor model. Importantly, these results also implicate increased T cellinfiltration caused by LSD1 inhibition as a likely mechanism underlyingthe synergism between LSD1 inhibition and PD-1 blockade. Taken together,the combination of LSD1 inhibition and PD-1 blockade may work throughsimultaneously eliciting anti-tumor adaptive immunity and reinvigoratingdysfunctional T cells to achieve a synergistic effect for tumortreatment. Given the general role of LSD1 in regulating dsRNA andinterferon responses, targeting LSD1 in combination with anti-PD-(L)1may prove to be a broadly applicable new strategy in cancerimmunotherapy. Furthermore, LSD1 inhibition may overcome the resistanceof B16 tumors to PD-1 blockade by increasing immunogenicity.

Example 10. Anti-PD-1 Treatment of B16 Tumors

To determine whether the synergistic effect between LSD1 inhibition andPD-1 blockade relies on the dsRNA sensor MDA5, immunocompetent C₅₇BL/6mice are anesthetized with Avertin (2.5%), shaved at the injection site,and then these mice are injected in the flank subcutaneously with250,000-500,000 B16-F10 tumor cells of scramble, LSD1 KO or LSD1/MDA5DKO (20 mice per genetically modified tumors, 20×3=60 mice in total).Tumors are measured every 2-3 days once palpable (long diameter andshort diameter) with a caliper. Tumor volume is determined using thevolume formula for an ellipsoid: 1/2×D×d² where D is the longer diameterand d is the shorter diameter. Mice are sacrificed when tumors reached 2cm³ or upon ulceration/bleeding.

For antibody treatments, mice are given 100 μg antibodyintra-peritoneally when tumor size reaches 200 mm³ or at day 8-10.Antibody treatment is repeated every other day for a total of fourinjections. The following antibodies are used: half mice receivinganti-PD-1 (clone 29F.1A12) are provided by G. Freeman (Dana FarberCancer Institute, Boston, MA) and the other half mice receiving ratIgG2a isotype control antibody are purchased from BioXCell (cat#BE0089). Prior to treatments mice are randomized such that treatmentgroups have similar average tumor volumes prior to treatment initiation.

Example 11. LSD1 Chemical Inhibitor in Combination with Anti-PD-1Treatment for B16 Tumors

To assess the efficacy of LSD1 chemical inhibition in combination withanti-PD-1 treatment in controlling B16 tumor growth, WT B16 tumor cellsare inoculated as described in Example 10 (40 mice in total: 10 mice forvehicle+isotype; 10 mice for GSK2879552+isotype; 10 mice forvehicle+anti-PD-1; 10 mice for GSK2879552+anti-PD-1). For inhibitortreatment, GSK2879552 is orally administrated or intra-peritoneallyinjected 1.5 mg/kg daily or every other day starting from the second dayafter tumor inoculation. For antibody treatment, anti-PD-1 or isotype isintra-peritoneally injected very other day starting from day 8 aftertumor inoculation for a total for four injections. Tumor volume isrecorded and is determined using the volume formula for an ellipsoid:1/2×D×d² where D is the longer diameter and d is the shorter diameter.Mice are sacrificed when tumors reached 2 cm³ or uponulceration/bleeding.

Example 12. Syngeneic Tumor Models with LLC, D4M and Renca Cells

To determine whether these findings can be generalized to other tumormodels, LSD1 is deleted by CRISPR/Cas9 LLC cells, D4M cells and Rencacells. LLC LSD1 KO cells, D4M LSD1 KO cells and Renca LSD1 KO cells(250,000-500,000 per mouse) are injected into their syngeneicimmunocompetent mice (LLC to B6 mice, D4M to B6 mice and Renca to Balb/cmice). Tumors are measured every 2-3 days once palpable (long diameterand short diameter) with a caliper. Tumor volume is determined using thevolume formula for an ellipsoid: 1/2×D×d2 where D is the longer diameterand d is the shorter diameter. Mice are sacrificed when tumors reached 2cm³ or upon ulceration/bleeding.

Example 13. WT Versus LSD1 KO B16 Tumor Growth in B6 Mice forTumor-Infiltrating Lymphocyte (TIL) Analysis

To analyze the tumor immunogenicity ant anti-tumor immunity caused byLSD1 deletion, B16 tumor cells are inoculated into immunocompetent miceas described in Example 10 (5 mice for WT control and 5 mice for LSD1 KOB16 cells). At day 12, mice are sacrificed and tumors are collected.Isolated tumors are then excised into small pieces and are digested bycollagenase to obtain single cell suspension. Some of the cells aredirectly stained with appropriate antibodies for profiling variousimmune cells, including CD4 T cells, CD8 T cells, macrophages, DCs andNK cells. Some of the cells are fixed, are permeabilized and are stainedwith anti-TCRβ, anti-CD4, anti-CD25 and anti-Foxp3 for Treg cells, andanti-TCRβ, anti-CD8 and anti-GzmB for effector CD8 T cells. In addition,some of the cells are re-stimulated for intracellular cytokine staining,such as IFN-γ and IL-2. Stained cells are subjected to flow cytometryfor analysis. Alternatively, some tumor samples are subjected to IHCanalysis.

Example 14. WT Versus LSD1 KO B16 Tumor Growth in B6 Mice forTumor-Infiltrating Lymphocyte (TIL) Analysis

To analyze the tumor immunogenicity and anti-tumor immunity in thesetting of LSD1 ablation plus anti-PD-1 treatment, B16 tumor cells areinoculated into immunocompetent mice, which previously would havereceived anti-PD-1 or isotype antibody injection at day 8 and day 10 (5mice for scramble B16+isotype; 5 mice for LSD1 KO B16+isotype; 5 micefor scramble B16+anti-PD-1; 5 mice for LSD1 KO B16+anti-PD-1). At day12, TIL are analyzed as described in Example 13.

Example 15. B16 Tumor Growth in WT or IFNAR1 KO Mice

To determine if B16-derived IFN-β is important for LSD1 deletion-inducedanti-tumor immunity and what types of cells are the crucial targets ofIFN-β, B16 tumor cells are inoculated into immunocompetent WT or IFNAR1KO mice, which would receive anti-PD-1 or isotype antibody injection atday 8, 10, 12 and 14 (5 mice for scramble B16+isotype; 5 mice for LSD1KO B16+isotype; 5 mice for scramble B16+anti-PD-1; 5 mice for LSD1 KOB16+anti-PD-1; 5 mice for LSD1/IFN-β DKO B16+isotype; 5 mice forLSD1/IFN-β DKO B16+anti-PD-1, 5 mice for LSD1/IFNAR1 DKO B16+isotype; 5mice for LSD1/IFNAR1 DKO B16+anti-PD-1). Tumors are measured every 2-3days once palpable (long diameter and short diameter) with a caliper.Tumor volume is determined using the volume formula for an ellipsoid:1/2×D×d2 where D is the longer diameter and d is the shorter diameter.Mice are sacrificed when tumors reached 2 cm3 or uponulceration/bleeding.

Example 16. Translational Significance

To demonstrate that these findings have translational significance, thepublic datasets on human cancer were explored. LSD1 was infrequentlymutated, amplified or deleted in a majority of cancer types examined(FIG. 12A), but LSD1 was found to be overexpressed in cancerous tissuescompared with normal tissues in a variety of cancer types (FIG. 12B). Todetermine whether LSD1 expression level in tumors correlated withclinical outcome, patients of each cancer type were divided by LSD1expression median, and overall survival between the two groups wascompared. This analysis showed that LSD1-high group had a significantlyshorter overall survival time than LSD1-low group for a number of cancertypes (FIG. 12C), suggesting LSD1 overexpression is a poor prognosticfactor. In line with the finding that LSD1 inhibition causedIFN/antiviral response in in vitro MCF-7 cells and ex vivo B16 cells(FIGS. 1N and 10J), LSD1 expression level was found to be inverselycorrelated with IFN/antiviral response in a variety of cancer types inTCGA cancer patient dataset (FIG. 12D). LSD1 expression level was alsoinversely correlated with CD8⁺ T cell infiltration in most cancer types(FIG. 12E), consistent with the finding of increased T cell infiltrationby LSD1 inhibition in mouse models (FIGS. 10A and 10P).

Further analysis on the TCGA skin cutaneous melanoma (SKCM) cohortshowed that patient group with low LSD1 expression (LSD1-low) had bettersurvival probability than that with intermediate or high LSD1 expression(LSD1-int/high) (FIG. 12F), and consistently, LSD1-low group wasassociated with increased expression of genes enriched in immuneresponses (FIG. 12G). Specifically, both CD8a and GzmB were expressedhigher in the LSD1-low group than in the LSD1-int/high group, indicatingincreased CD8⁺ T cell infiltration (FIGS. 12H and 12I).

OTHER EMBODIMENTS

It is to be understood that while the disclosure has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of thedisclosure, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

1. A method of treating cancer in a patient, the method comprising: administering to a patient in need thereof a therapeutically effective amount of a lysine-specific demethylase 1A (LSD1) inhibitor, to thereby treat cancer in the patient.
 2. A method of treating cancer in a patient, the method comprising: administering to a patient in need thereof a therapeutically effective amount of a lysine-specific demethylase 1A (LSD1) inhibitor and at least one immunotherapy, to thereby treat cancer in the patient. 3-6. (canceled)
 7. The method of claim 2, wherein the at least one immunotherapy is selected from the group consisting of: an antibody, an adoptive cellular therapy, an antibody-drug conjugate, a toxin, a cytokine therapy, a cancer vaccine, and a checkpoint inhibitor wherein the checkpoint inhibitor is a CTLA-4 inhibitor, an OX40 inhibitor, a TIM3 inhibitor, or a LAG3 inhibitor.
 8. (canceled)
 9. The method of claim 1, wherein the LSD1 inhibitor is selected from the group consisting of: a small molecule, an antibody, and an inhibitory nucleic acid.
 10. The method of claim 9, wherein the LSD1 inhibitor is an inhibitory nucleic acid, and wherein the inhibitory nucleic acid is a small interfering RNA or a short hairpin RNA.
 11. The method of claim 10, wherein the inhibitory nucleic acid is a short hairpin RNA and the short hairpin RNA comprises SEQ ID NO:
 2. 12. The method of claim 9, wherein the LSD1 inhibitor is a small molecule selected from the group consisting of: tranylcypromine, RN 1 dihydrochloride, GSK-LSD1, GSK2879552, ORY1001, GSK690, namoline, Cpd 2d, S2101, OG-L002, SP2509, CBB2007 and IMG-7289. 13-20. (canceled)
 21. The method of claim 1, wherein the cancer is a primary tumor.
 22. The method of claim 1, wherein the cancer is a metastatic tumor.
 23. The method of claim 1, wherein the cancer is selected from the group consisting of: melanoma, acute myeloid leukemia (AML), squamous cell carcinoma, renal cell carcinoma, non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), gastric cancer, bladder cancer, kidney cancer, head and neck cancer, Ewing sarcoma, Hodgkin's lymphoma, Merkel cell carcinoma, breast cancer and prostate cancer.
 24. The method of claim 1, wherein the cancer is a non-T-cell-infiltrating cancer.
 25. The method of claim 1, wherein the cancer is a PD-1 and/or PD-L1 refractory cancer.
 26. The method of claim 1, wherein the cancer is a PD-1 and/or PD-L1 resistant cancer.
 27. The method of claim 1, wherein the patient has previously received a cancer treatment.
 28. The method of claim 1, wherein administering occurs at least once a week.
 29. The method of claim 1, wherein administering is via intravenous, subcutaneous, intraperitoneal, rectal, and/or oral administration. 30-32. (canceled)
 33. The method of claim 1, wherein the method further comprises administering a chemotherapeutic agent to the patient.
 34. The method of claim 1, wherein treating comprises reducing the volume of primary tumor in the patient.
 35. The method of claim 1, wherein treating comprises delaying cancer progression in the patient.
 36. The method of claim 1, wherein treating comprises modifying the tumor microenvironment of a cancer in the patient, sensitizing cancer to a checkpoint inhibitor therapy, decreasing the risk of developing at least one metastatic tumor in the patient, decreasing the rate of tumor growth in the patient, and/or eliciting tumor-intrinsic double-stranded RNA stress in a cancer cell in the patient. 37-40. (canceled)
 41. The method of claim 1, wherein the method comprises identifying the patient as being resistant to PD-1 inhibitor or PD-L1 inhibitor treatment.
 42. The method of claim 2, wherein the method comprises identifying the patient as being resistant to PD-1 inhibitor or PD-L1 inhibitor treatment. 